Lecture 13. Protein Function and Enzymes.
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Weiss, Gregory Alan
 Introduction to the basic principles of chemical biology: structures and reactivity; chemical mechanisms of enzyme catalysis; chemistry of signaling, biosynthesis, and metabolic pathways. 
 We're going to pick up where we left off. Hopefully you watch the podcast lecture on Tuesday. I want to pick up what we discussed on Tuesday, looking at protein function. Specifically, we're going to be talking about how enzymes work. How do enzymes catalyze key reactions in the cell and so on. We're going to start by talking about some measures of enzyme activity. And then we'll talk about regulation of enzymes. And then we'll get into the mechanisms. And then we'll close with mutagenesis and engineering. So what we talked about on Tuesday is that proteins have a wide range of roles, structural, binding, and catalytic roles. In the example of structural, we saw, for example, collagen. We saw how collagen gets organized into these complex assemblies that make it possible to have extremely strong bones and things like that. We also talked a little bit about Titan, a muscle protein that makes muscles capable of being pulled and stretched without breaking, without snapping. We also talked about binding. The example we saw there was FKBP binding to FK506 and rapamycin. And then finally, we're now up to the part where we can start talking about catalysis. So protein function has at least three major roles inside the cell. Structure, binding, and catalysis. And when we talked about the repeat proteins, for example, we saw a great example of binding. And I want to just very briefly, we'll take a look at that at a moment, but I just want to emphasize something based on some questions that I got during my office hours. We talked about how noncovalent receptor ligand interactions can be described by dissociation constants and an on-rate and an off-rate. We're now at the point where we're going to start talking about this, Michaelis-Menten constant, KM, which is an analog to KD. And something that I touched on at the very end of the lecture is enzymes work by lowering the transition state enzyme of the reaction, energy of the reaction. Doing this is in essence how you catalyze a reaction. So lowering the activation energy necessary for that reaction to take place, you're increasing the speed that the reaction takes place by doing this. And enzymes do this by binding to the transition state and stabilizing it. By having counter ions that stabilize charge functionalities in the transition state, the enzyme can lower the transition state energy. And this is surprisingly effective. We're going to be seeing some examples of that today. So we're going to be talking today also about how this catalysis by enzymes is coupled to the motion by the enzyme. And I'll show you some examples of this. And what we're going to see is that enzymes force the substrate, the starting material, into confirmations that favor formation of the product. And by doing that, that accelerates these reactions. Okay, and very briefly, I just want to touch on one point about the repeat proteins. Something I didn't mention but probably should have when I talked about the repeat proteins. This is an example of a repeat protein. This is an anchorin repeat. Notice that it has a series of helix, turn, helix, turn, helix, turn, helix, turn, helix, turn, helix, turn, helix. So those repeats, each one of these loops and then helix, loop and then helix. And notice that they're all stacked on top of each other. Each one of those is one example of a repeat. And this anchorin repeat has a whole bunch of these lined up. Okay, that's where it gets the name repeat protein. Make sense? And then similarly with the leucine rich repeats, in this case, it's a helix, strand, loop, helix, strand, loop, helix, strand, loop, et cetera. Okay, but it's a series of repeating motif, repeating structural motif. In fact, these are actually individual domains. They fold modestly well on their own and they can be shuffled around to a limited extent. Okay, any questions about the topic of protein structure? And for that matter, any questions about anything that was covered on Tuesday? In Tuesday's lecture. All right, well I think in that case then we're ready to move on. We're going to talk next about catalysis. Today's discussion is going to be pretty high level. I'm going to be telling you stuff that's actually not in the book. And in fact, actually it's only found really in the frontiers of the literature in chemical biology. So don't hesitate to interrupt if at any point I start to lose you. Okay, it's better if you interrupt me early on than if I go further down the road and then you're totally lost. Okay, because the truth is this lecture will be the only time that you're going to be able to find this material I'll be discussing. And I think it's very, very important. It's actually the very frontiers of chemical biology. Okay, so I want to talk to you today about catalysis. And last time I showed you that non-covalent binding consists of ligands hopping on to some binding site in a receptor. And we describe this non-covalent interaction using an equilibrium constant, a special equilibrium constant called a dissociation constant that quantifies the ratio between unbound up here to bound receptor ligand interactions. Okay, so this is pretty straightforward stuff. The only difference with catalysis is that we're going to have this similar receptor ligand interaction. But upon binding to the ligand, the enzyme in this case instead of the receptor, and for that case instead of the receptor, the ligand is going to be transformed, converted into some new product. So it's a similar sort of process. So if we understand non-covalent interactions, then we can also understand catalysis by enzymes. Okay, I'm skipping ahead, skipping, skipping. We talked about this already. All right. This is, I believe this is where we left off, right? Is this right? Okay, so where we left off again is this idea that receptor ligand interactions are governed by some binding. In the case of enzymes, the substrate binds and then some catalysis takes place to convert the substrate into a product. S stands for substrate or starting material, and that gives us P, which is the product, and then the product has to dissociate. I think I've talked about this, right? This was covered. Right? I did cover this. Okay, good. Right, okay, so I think I'm actually over here. Okay, good. So here's a typical reaction scheme I already talked about on the previous thing. So the formation of this enzyme substrate product, this mechalosmentin complex, this is sometimes called the mechalosmentin complex, this complex here of the activated enzyme bound to the substrate in a way to catalyze the reaction has an equilibrium constant, and in the same way that the KD was proportional to the rate constants for the dissociation, the K off to K on, in the same way the mechalosmentin equilibrium constant is equal to the sum of the off rate and the K cat. So that's like getting to the ES and either going backwards or forwards. That's the rates going in either direction for destruction of the complex versus forming the complex, meaning the rate constant for K on. This KM resembles the KD for non-covalent binding interactions, and so it's useful for us. The strength of the KM tells us something about how avidly the enzyme is going to grab onto the substrate and how quickly it's going to form this ES complex. So that, this KM is actually a useful number. It tells us something about the conditions inside the cell because the enzyme has to evolve up to a sufficient affinity for its substrate so it can grab onto the substrate inside the cell. If the affinity is too low, then the enzyme substrate complex will never form and the enzyme will never catalyze a reaction. On the other hand, if it evolves to the point where it's super-duper high, maybe that's not so useful for the cell because maybe the enzyme then will be a little too active. So these evolve up to the maximum ability that's necessary for the cell. That's required for the conditions found inside the cell. And oftentimes, for example, we can engineer enzymes to have very different KMs simply by tinkering with their active sites. And I'll talk about that more in a moment when we talk about protein engineering. Okay, so if we look at a dose response diagram, this is similar to the dose response diagrams that I showed on a previous slide on Tuesday. Instead, on the y-axis, we have our initial reaction velocity and the concentration of the substrate, the point of inflection here is going to be roughly the KM, this equilibrium constant up here. For that matter, this point of inflection also tells us where 50% of the maximum rate of the enzyme is going to be. At the very highest concentrations of substrate, the enzyme is going to be running flat out. Okay, so that's like as fast as the enzyme can possibly go. It would be the equivalent of giving the sprinter maximum oxygen, maximum glucose. Everything he or she needs to do to run as fast as possible. So that's up here under maximum velocity conditions. Notice that this asymptotically approaches this V max value way up here. Okay, and so anything where it's an excess concentration of substrate that's called V max conditions and typically enzyme reactions that are run in the laboratory are run under those conditions. We always have an extreme excess of substrate typically. Okay, let's take a look at some KMs. They range widely. There's a wide range of possible KMs for enzymes. And here are some numbers over here. Now, like the KD, a lower KM value means tighter binding. Totally analogous to the dissociation constant. In fact, it has a very similar connotation. So what this tells us, for example, is that this enzyme here, cytochrome P450, binds benzopyrene with theory high affinity. This should come as no surprise to us. Benzopyrene is this big, flat hydrophobic molecule. And hydrophobic molecules in general aren't so soluble. So the cytochrome P450 in your liver is going to be grabbing on to the benzopyrene that you inhaled on your way to over here when you got behind that stupid shuttle bus that was, you know, dinking along at too slow a speed. Right? So you get behind the exhaust pipe of that shuttle bus and you start inhaling unburnt benzopyrene. So this cytochrome P450 in your liver is right now, as we're speaking, grabbing on with great affinity for these benzopyrates. On the other hand, there are some enzymes that don't have to grab on all that well to their substrates like aconitase. This is a key enzyme in the metabolism of glucose. And its substrate, citrate, is found at high enough concentrations that the enzyme doesn't really have to evolve to a very high affinity. So this gives us sort of a crude measurement of what the concentration of the substrate is in the cell. Right? So what we know is that there's probably not a lot of benzopyrene present, but there's probably tons of citrate present, hence the need for lower affinity. Now, let's also take a look at some K-cats. So this is the equal, this is the rate constant for the decomposition of the macalus menten complex, the ES complex that is now being broken down to form enzyme product. Okay? Make sense? Okay. So in this case, again, there's a wide range of K-cats. And this tells us something about how hard the reaction is to catalyze. Harder reactions in general have lower K-cats. But it can also tell us something about the evolution of the enzyme. Enzymes in general evolve up to the required function and really don't go past that. Okay? There's really no evolutionary drive. There's no selection mechanism that drives the enzymes to be perfect unless they need to be perfect for some particularly crucial function for the cell. So here's one example of a really crucial function for the cell. The enzyme catalyze breaks down hydrogen peroxide into oxygen and water. This is a crucial reaction. Hydrogen peroxide creates a substantial burden on cells. This is a strong oxidant. And oxidants run around and wreck havoc on cellular machinery. And so for this reason, cells have evolved pretty sophisticated mechanisms to very quickly break down such oxidation products. And catalyze has a K-cat of 100 million. So this is a really, really fast catalytic reaction that takes place. And then a slower reaction would be a protease. Protease is of course hydrolyzed amide bonds. I believe we've seen these before. And their K-cats are much lower likely because this reaction is a little more challenging and a little less favorable thermodynamically. And also for that matter, it's not as critical perhaps for the cell. Okay. Questions so far? Good. Okay. So these are the numbers that are going to underlie our discussion as we start talking about the properties of enzymes. Okay. These are the same numbers that you learned about in like Bio 99 or 98, whatever biochemistry class you take here at UC Irvine or elsewhere. These numbers are kind of the vocabulary that my biochemistry friends talk about when they talk about enzymes. Okay. Now the truth is as a chemical biologist, I don't get too worked up about these numbers. I'm more interested in understanding the atoms and bonds basis for how the enzymes work. And so I guess the best place to start would be let's start with the perfect enzyme. What would be the enzyme that really can crank, that could maximize its ability to turn over a reaction? And then we'll look at some specifics at the level of atoms and bonds. So the very perfect enzyme, you might imagine, every time it forms this Michaelis Menten complex, the ES complex, then it goes immediately to Kcat. So it forms and then boom, it's over to the Kcat. And it just immediately gets converted, converts the substrate to the product that happens instantly. On the other hand, the perfect enzyme is not going to have any off rate over here. This off rate represents lost opportunities. This is the sea, the wasteland, coulda, shoulda, maybe shoulda. Okay, right? This is the chance that the enzyme missed. So instead of going to product, the enzyme goes backwards. And so this off rate over here is miserable and inefficient for an enzyme. So the perfect enzyme is not going to have an off rate. And so the perfect enzyme, you can basically imagine Koff being 0. And if we have that, then we can imagine rearranging our KM equation shown a couple of slides ago, such that Kon equals the ratio of Kcat to Km. And again, notice that these little k's are indicating rate constant and the big Km is for equilibrium constant. So the very best enzyme will have an on rate that's diffusion controlled. In other words, it's limited by the amount of time that the substrate, Brownian motion style, eventually bounces its way to the active site. That should be the slowest step for an enzyme that's perfect. And we've talked about this before, but that rate of diffusion has a speed limit of 10 to the 9th per molar per second. It can't go any faster than that. That's a physical law. It's like the speed of light. You cannot exceed that. Just because it takes a little while to bounce around through all that water and other stuff that's present in the cell. Okay, make sense? We'll take a look at a moment at an example of an enzyme that's far from perfect and we'll start to understand what its sources of imperfection are. So before we do, let me just give you a little table that I really like that shows us and helps us organize enzymes. This shows us the rankings of enzymes in your proteome. Okay, so this is a listing from most common to least common. It's like a greatest hits of the seven categories of enzymes that are found in the human proteome. The most common enzymes by far are the hydrolysis. These are the enzymes that introduce water as a way of breaking a bond and we're going to see a couple of examples of this. We'll see examples of glycosylases and proteases today. We've already seen examples of nucleases. That was stuff like RNAase, right? Remember when we talked about RNAase and it was inhibited by Dipsy? This is a similar sort of thing. Can someone help this guy out? Thank you. Okay, transferases, next most common, second position. These are examples of enzymes that transfer functionality from one spot to another and we're going to look in detail at an example of a protein kinase today and then later in the class we'll look at a glycosyl transferase. Oxido reduptases, this is like the enzyme cytochrome P450 that takes the benzopyrines, introduces an epoxide and oxidizes the benzopyrines. You all remember this, right? I showed you the benzopyrine a couple of slides ago but earlier in this quarter when I was talking about cigarette smoking, I showed you how the benzopyrine that looks like this rather innocuous flat structure gets converted into an epoxide and then slips into the pi stack of your DNA and alkylates the DNA. And so these are actually very common enzymes, these oxido reductases because they're important for removing toxins. Dehydrogenases are another one that's very common and perhaps we'll get a chance to see this one today. And then finally we get down to the ligases. These are enzymes that spot weld together to functional groups such as attaching ubiquitin or DNA to something. Dysomerases are used to convert substrate into some related isomeric product. These are things like epimerases. The synthetases we've seen before, we talked about aminoacyl tRNA synthetase. This was that gargantuan complex that read out the anti-codon and the various modifications of the tRNA to make sure that the correct amino acid was being attached to the tRNA during aminoacyl tRNA synthesis. Okay, so and then the final one, the liases are doing things like decarboxylation. These are actually breaking carbon-carbon bonds in dramatic fashion. So these are aldolases that are doing aldol reaction, et cetera. They're either breaking or making carbon-carbon bonds. So fairly, I feel like we've seen many examples of these different enzymes this quarter. So now I can go through and just talk about the ones that are really important that we haven't seen yet, okay, such as the kinases over here. And I believe that's where I'm going to start. Yes, in fact it is. So it turns out that kinases have a common fold that consists of a lower domain down here and then a larger lobe up here. The active site is indicated where these Van der Waals spheres are. This is ATP. So kinases take ATP and transfer the gamma phosphate of ATP to some sort of hydroxide recipient. Okay, that's generically what they're doing. When we talk about the gamma phosphate, ATP, adenosine triphosphate, ATP has three phosphate groups called alpha, beta, and gamma. The third one in the row is called gamma. And so that's the phosphate group that the kinases are going to transfer. So again, notice that these have a conserved dual lobe structure even though these have widely disparate activities. This is everything from a receptor tyrosine kinase over here to protein kinase over here. These do have very different activities. They phosphorylate different targets. And yet on the other hand, they all evolved to have very similar structures. Now, this class of enzymes, like all enzymes, can be inhibited by substrates, pseudo substrates that mimic the real substrate. So here's the structure of ATP, but in place of one of the oxygens of ATP highlighted in blue, we have a nitrogen. And this phosphoramide inhibits the kinase. Okay, so if you feed this phosphoramide to a kinase, to any of the kinases I showed on the previous slide, it's going to be game over for them. They're not going to be able to work because they're going to bind to this ATP analog. They're going to put it in a sloppy old beer hug, but this gamma phosphate is missing the oxygen. And missing the oxygen is the same as saying it's totally inert. And so this is going to basically be locked in the active site, in the brace of the active site, yet unable to transfer the phosphate group. And so the net effect is to shut down the enzyme. And this is a very effective way of killing enzymes. You basically know something about the mechanism. You make a tiny little modification of the substrate, and boom, it's game over for the enzyme. Okay, so you could do this also with sulfur or nitrogen. So I have shown you the nitrogen, but you could do that as well. And again, it sticks in the active site and inhibits the enzyme. This approach also works if you mimic the product. And a large number of enzyme inhibitors pursue one of those two approaches. Either mimicking the substrate, as shown here, or mimicking the product. Either approach works great. So let's zoom in. I showed you the structure, the bilobe structure of kinase. Let's zoom in and take a look at its active site. In the active site, there are, here's the structure of ATP. There are a series of conserved magnesium ions, these balls over here, that are bound to the phosphate groups of the ATP. The numbers here indicate an angstrom the distances. Okay, and these numbers are pretty low. Right, if you recall that a carbon-carbon bond is somewhere on the order of like 1.5 angstroms or so. These are pretty close in numbers, right? These, this magnesium is getting awfully close to this oxygen over here. These are cozy, cozy molecules and atoms. They like being this close. They like being this close because they have complementary charges, right? The magnesium has a plus 2 charge. The oxygens of these phosphate groups have negative charges. So they're attracted by salt bridges or coulombs interactions that we saw earlier in the quarter. So over here, there is the other substrate for this enzyme reaction that has a hydroxyl. I'm showing it to you with the hydroxyl deprotonated. And after a phosphoryl transfer, this oxygen of the substrate has now picked up a phosphate group. And notice that the magnesiums here are helping to stabilize that phosphate group, right? That they're lowering the energy of binding by forming that same sort of coulombic salt bridge that we saw earlier. Okay, now if we zoom in and take a look at the arrow pushing of, arrow pushing mechanism for this enzyme active site, what we find is that and not depicted on this previous slide, somewhere out here, there's a carboxylate side chain from aspartic acid. The carboxylate of this aspartic acid deprotonates the proton of the hydroxy of a serine for the substrate. And that sets us up with an alkoxide. Alkoxide being a superb nucleophile, it's negatively charged, can attack the gamma phosphate of ATP. And again, the magnesiums get in on the action. They're over here participating fully and stabilizing this negative charge of the phosphate group. That's crucial, right? You can imagine this reaction not going in the absence of those magnesiums, right? Because one negative charge is not going to want to approach a negatively charged phosphate group, right? The negatively charged alkoxide over here is going to be stymied in its attack. It's going to get repelled by this phosphate group. So the magnesiums are shielding the phosphate group and protecting it and preventing it from getting, from looking like a negative charge. And so that tees up this reaction very deeply. And then finally there's a collapse of this trigonal bipyramidal intermediate. And just very briefly, the structure around this phosphate looks like a trigonal bipyramid. Not so, anyway, that's interesting. And then there's collapse of this trigonal bipyramidal intermediate to give us our final product. Okay, so to summarize, the most important aspect of this is the notion that the magnesium ions are playing several roles to make this reaction possible. First, they're coordinating and stabilizing the transferred phosphate group as a Lewis acid. Okay, so that helps accelerate the reaction as a Lewis acid. Turns out that Kine's activity in the cell is very tightly regulated. And the reasons for this are perhaps not clear if you don't know much about signal transduction. And I'm just going to very briefly cover it today. Then in a future lecture, we'll learn quite a bit more about it. In the cell, there's a series of pathways that transfer information. And these pathways are controlled by transfer of Kine, transfer of phosphate groups to key residues and proteins. So Kine's play a really key role in kicking off various processes in the cell. So there's cascades of Kine's where one Kine's phosphorylates the next Kine's which phosphorylates the next Kine's and so on and so forth. Turns out that this process is very tightly regulated because you don't want your cells going wild. You don't want them to be doing uncontrolled cell division, for example. And so for this reason, the cell very tightly regulates Kine's activity. And I want to show you a couple of vignettes about this tight regulation because it's crucial to our understanding of how Kine's work. Okay, so here's one example. This is an example from the enzyme protein Kine's A, cyclic AMP regulated Kine's. And the way this works is there's actually a regulatory subunit shown here in blue that binds to the Kine's and actually has an inhibitory loop that blocks access to the active site. Okay, so does everyone see how this dark blue thing is? It's binding here and then it has like this long finger that fits into the active site and blocks the Kine's from binding to any substrates. This shuts down the Kine's and the ability to shut off the Kine's is crucially important. Okay, if you don't have this, the Kine's will be running around rampant wrecking havoc, turning on stuff, shutting off stuff, causing death and destruction and general mayhem. And I do mean death and destruction. Okay, these Kine's are that important. Now, when levels of a reporter molecule called cyclic AMP reaches certain concentration, this cyclic AMP binds to the regulatory subunit and causes the regulatory subunit to dissociate from the catalytic subunit approaching Kine's A. So these two molecules get forced apart as the blue one flips into a new confirmation. Upon binding to cyclic AMP, the thing changes its shape and it no longer has affinity for protein Kine's A. This is good, this is your protein Kine's A. It frees it up to go off and do the mission that it's wanted to do for its entire life, which is to run around the cell and phosphorylate anything that moves. Nearly anything that moves. It actually has a little bit of specificity, but for the most part, protein Kine's A likes to phosphorylate lots of different binding partners. Okay, this is a pretty promiscuous molecule. Now, here's the thing. Another way of regulating enzymes is to phosphorylate them. Okay, so this is one way where you have some regulatory protein that binds. A second way is to phosphorylate residues that are near the active site. Okay, so for example, this non-hydrolyzable analog of ATP, which has the nitrogen in place of oxygen. This is the molecule I showed on a previous slide. This tells us where the active site is. But over here are two residues that can be phosphorylated to flip on this map Kine's, this P38 gamma map Kine's. And so one of these is a tyrosine and the other one is a serine. And so this Kine's waits around until it gets to the, until it gets phosphorylated and at that point it goes into gear. Okay, so this is like an on-off switch for the Kine's. In the absence of this, the enzyme doesn't have the right confirmation. It doesn't have the right confirmation. It can't be a Kine's. Okay, so the phosphorylation of the Kine's puts it in gear, turns it on, and sets it going. Does this make sense? Any questions about what you've seen so far? Okay, that's the basics. I want to talk to you about the really neat stuff, the latest results in thinking about how Kine's is work and thinking about their motions. And again, this is kind of an abrupt departure from sort of standard material as presented in biochemistry classes. And it really represents the frontier in chemical biology. Many of the next experiments I'll be talking about were done, actually, with Miriam. She's one of the leaders in this area. Okay, so the thing is I want to talk to you about how enzymes work at a mechanistic level and how they actually work dynamically. How do they move when they do these reactions? So I should tell you that enzymes have great motions associated with their activities. This is unlike the case of conventional catalysis by organometallic complexes that you learned about back in Chem 51, okay? Or if you learned about in Chem 125. In those cases, the organometallic catalyst binds and perhaps it plays some Lewis acidic role, but it's certainly, we don't think about its motion. Okay, we don't think about it having some, you know, movement associated with it, some dynamics. Enzymes, it turns out, for the most part, almost all have very wild and very quick motions associated with them and a frontier in chemical biology is to understand how those motions impact catalysis. How do those motions allow enzymes to be effective catalysts? And so it turns out that if you get a big, you know, round bottom flask full of enzymes, you'll never be able to see those individual motions. And the reason is they tend to get blurred out, okay? So if we look at a large number of molecules, we'll never see the individuals in motion because all of the enzymes in that flask are going to be running along at different speeds and everything gets blurred out. Okay, so instead, in order to see individual motions, we have to look at single molecules. And to understand this a little bit better, let me offer an analogy. Let's imagine that I convinced, you know, the Orange County Marathon folks to reroute the marathon. So instead of being on Sunday morning, instead I convinced them to run it here Thursday at 10, 10 a.m. And I convinced them to start at that end of the classroom and send all the runners through the classroom from that door to this door. Okay, so now everyone's running through here all 10,000 runners. You can imagine what you're going to see is just a blur of pumping arms and legs, right? Everyone's going to be trying to get in and out of this classroom as quickly as possible. It's going to be total pandemonium. That's the situation when we look at an ensemble of enzymes. It's total pandemonium. It's a blur of arms and legs. We don't see anything. Everything gets averaged out. And by see, I mean using tools like spectroscopy, using tools that you're familiar with from your other classes. So over the last 15 years, there's been a revolution in this area of chemical biology or biophysics where scientists have started to look at individual molecules in isolation from all of their other friends and neighbors. Okay, so now instead of having the entire marathon coming through the classroom, let's imagine that I convinced each runner to come running through one at a time. So they're going to start over there and then come running through here. What you will see because each runner is isolated from all the other runners is you'll be able to see their arms and legs moving, right? Because now there's no blurring out effect, right? And furthermore, if you look closely, you'll be able to see some runners moving faster than others. Maybe one runner has a different stride than her neighbor, right? Because she comes running through and I don't know, maybe she extends her leg a little longer than the runner behind her. Okay, but if we convince them to be isolated from each other, then we can really start to get information about how they move. And that's the situation we find ourselves in when we start looking at enzymes. And so this area of single molecules allows us to look at conformational steps, at intermediates, and the kinetics and dynamics that underline enzyme function. And this again is a major frontier and it's a really exciting area to be involved in research. Okay, so everyone with me so far. Everyone understands this idea of looking at single molecules, right? Okay, good. I want to talk to you next about how we're going to observe our single molecules. There are a number of different fluorescence techniques for looking at single molecules. Patch clamping you may have heard of is a 40-year-old proven technology that works really well for looking at individual receptors. That works fine. In the last five years or so, groups here at UC Irvine have been at the forefront for inventing sort of a tiny little microphone that allows us to listen in to enzymes as they run. And it has some advantages over those other techniques. And so that's what I want to talk to you about today. Okay, so this is a collaboration between my laboratory and Phil Collins in the Department of Physics here at UC Irvine. And he's pioneered ways of building circuits that are based on carbon nanotubes. And this is an example of a carbon nanotube. This is basically a carbon graphite graphene layer that's folded up into a cylinder. It looks sort of like chicken wire. These wires though are amazingly conductive. Carbon nanotubes are really remarkable material. They have remarkable mechanical properties. They have remarkable properties for conducting electricity and for conducting heat. In terms of conducting electricity, all of the electricity is going to be flowing through the outside of the wire, through these bonds out here. And the electricity is not flowing through the middle of the wire. So this is unlike, for example, the copper wires that are used in wiring the walls, okay, wiring the electrical outlet over there on the walls. And this property makes the outside of the wire superbly sensitive to tiny little perturbations on its surface. And that's what we're going to do. So here's the way we do this. Students in the Collins laboratory and my laboratory start with silicon wafers that are about this big. We go to the engineering building across campus. The students put on bunny suits. And we build using photolithography circuits that look like this. There are these contact pads to which we attach wires. And then down here, you get down to these interdigitated electrodes that do not touch each other. So this is an open circuit. But somewhere out here, we sprinkle an iron militant of catalyst that catalyzes growth of one of these carbon nanotubes, a single walled carbon nanotube, across the wires to complete the circuit. And I false colored it in red over here. Okay. So now what we do next is we turn this wire into the world's tiniest microphone. We turn it into a device called a field effects transistor. It's not so important how that works. What matters is it's more or less the same as the microphone found in my cell phone. Okay. Same principle. And next, we're going to glue individual proteins directly onto the microphone and listen as they flap around. Okay. So runners, if we had runners running through here, you'd expect to hear the pounding of their feet, right? And you'd expect to be able to interpret the noise of their feet to tell us something about their stride, whether or not they're accelerating, whether or not they're slowing down, et cetera, whether or not they have funny heel strike, et cetera. Right? Makes sense? So we're going to do the same thing but with proteins. Now, I know that you're probably thinking proteins don't have noise. I have proteins all over my body and I'm not hearing anything right now. And the truth is the noise is very, very tiny. It is so tiny that it's very hard to hear. But moving charges do make noise. And I'll give you one example of this. If you're at the beach with a bonfire down here at Little Corona Beach State Park and you have this big bonfire going, you know how the wind kind of whips the flames and the flames make this kind of neat, whooshing noise? That sound of the flames moving is due to plasma in the flames that's charged ions in the flames that are moving around. So charged functionalities make noise as they get pushed around. And in fact, actually there's a speaker, a loud speaker. These are really expensive stereo speakers. On the order of like $20,000 a pair. They better sound good at that price. But it's actually based upon having a plasma that's moved around by a little magnetic coil. Okay? So you can actually hear charged ions moving around. And that's what we're going to do when we glue in the protein. So let me show you what it looks like when we have the protein glued in. This is the schematic diagram over here. We have the carbon nanotube. Here's the protein glued in. This protein is streptavidin. Familiar, right? Everybody in this classroom. And we have streptavidin conjugated to a tiny little dot of gold and that's shown here. Okay? So the little dots here, that's the gold attached to streptavidin. And then the horizontal lines are the wires, the carbon nanotubes. And the vertical are the electrodes over here. And you can see we're getting one, one, one, one, one attachment. Okay? So the breakthrough that Phil and I came up with, with our coworkers, our friends, the graduate students, was that we developed a way of attaching one and only one attachment each time to the carbon nanotube. This means then that we're isolating the enzymes away from all their buddies, which means then we can start looking at confirmations and intermediates. Okay? This is all a long introduction. We're going to get back to the kinases in a moment. Before I do, let me just set the stage. Here's the experiment again. We have the electrodes. We have the carbon nanotube. It turns out that, of course, you can't run your cell phone in water, so you can't run one of these tiny microphones in water. Okay? I think I did this experiment last week. I dropped my phone in a bucket of water. Actually, it was my cat's water bowl. And I pulled it out quickly enough, but it definitely did cause some damage. So electronics and water don't mix. I don't think it surprises anyone in the classroom. And so for what we have to do is we have, but all biology, of course, takes place in water. So this creates a dichotomy. And to solve this, what we do is we cover up all the electronics with a layer of polymethylbethacrylate. This is shown here in gray. And then we blast a little tiny window using something called an electron beam that just opens up a little tiny region of the carbon nanotube. And that's where we're going to do the experiment. Now all the images I've been showing you up till now are electron micrographs using electron microscopy. We're now going to get really small as we start imaging individual molecules of proteins. This is so small that you can't really see them very readily using electron microscopy, except if you use that trick that I showed on a previous slide where you coat things with gold, that was just streped out of the thing. So now to see these things, we now are going to have to use atomic force microscopy, where we're now getting down to really tiny resolutions of one nanometer or so. So here's what it looks like. This is one enzyme attached to the carbon nanotube. And this is an AFM image, atomic force microscopy image, just showing the windowed region, just showing the carbon nanotube that's exposed. This little blob right here is actually the enzyme attached. It has the right dimensions for that one enzyme. And now we turn on the microphone and it's lights camera action. Okay, at that point then we're ready to listen in. Okay, one more image. This is before and then that's after. And where it's circled, you can see very clearly the enzyme attached. Okay, everyone's still with me. Questions so far? All right, yes? What are the other? Oh, thanks for, yeah, thanks for these other blobs that are kind of like, those are other little enzymes that we can't get rid of. It's actually enormously hard to do these sort of images. It just turns out the proteins are kind of sticky. There might even be some salt crystals somewhere out here. So there's always some garbage-y stuff that we've been totally unable to get rid of despite a lot of work. It took a lot of work to even get images that are this clear. So. Could it be different when an enzyme gets separate from others the way they add the react and the ray in some? Okay, that's a good question. Okay, that's a really good question actually. Thanks for not asking that. Thanks for not being on the study section that asked that. Okay, so the question is, would an enzyme in isolation from itself behave differently than an enzyme that's next to its neighbors, right? In the same way that a crowded field of runners is going to run differently than if a solo runner. We don't know. I would like to think it's going to run the same. But it is a legitimate caveat and I thank you for that. I will have to think about that some more. Thank you. So why do we study that? Why are we not in fact, you know, well-increased in that? Yeah, so enzymes frequently aren't really, so it's true. Enzymes are really crowded conditions inside the cell. But it's not like there's like a thousand enzymes that are all doing the same thing crowded together. It's more like there's a couple enzymes that are kind of jammed in with hundreds of other molecules inside the cell. You know, so they're not all doing the same thing. So we can recreate that sort of thing. We can recreate the crowded conditions inside the cell and compare. We haven't gone into the experiment, but I'd love to do it. So, okay? Okay, let's get back to kinases. So again, this is protein kinase A. It still has the two lobes that I showed earlier. Here's the big lobe down here and here's the smaller lobe up here. And somewhere close to the, this upper lobe, Miriam, together with Issa Moody, a former graduate student in the laboratory, engineered a single cysteine. The cysteine, of course, has a sulfur functionality, a thiol functionality, and that allows us to attach site specifically the enzyme to the nanotube, to this microphone down here, through a pyrene. Okay, so pyrene is making yet another cameo in today's lecture. And as you might expect, the pyrene is going to py-py stack onto the carbon nanotube. Okay, because it's so hydrophobic, it's looking for a nanotube to stick onto. And it turns out the enzyme is very firmly held in place. This is like a very special kind of molecular glue that sticks these two molecules together. But notice that it's being held in a non-covalent interaction. And in practice, this thing is held in place for 10 or 12 hours. We don't see it coming off. It's really stuck in there very firmly. Okay, and again, here's another AFM image. And that little blob attached to the carbon nanotube is our enzyme. Okay, yeah, question over here. Sergio. How do we ensure that we have just one enzyme attached and not two in the liquid? Yeah, so we do AFM, this technique of atomic force microscopy, before we get started with the experiment. Just to make sure that we have one attachment. And we use this special technique called in liquid AFM. If we see two attached, we wash it away, and then start over. If we see zero attached, we start over with attachment. Okay, yeah, question over here. So when you have one and you use AFM, I'm sure you have one. Yeah. You know, like for example, a second one doesn't attach afterwards when you start listening. Okay, so afterwards, we don't add any more enzyme. So we have like purified buffer that has no enzyme around. So, and I know what you're thinking. You're wondering, well, what about this blob over here? What if it decides to get up and wander over here? It turns out these blobs are pretty firmly stuck down on the surface. And another thing is if another one attached, we'd hear that one running alongside it. Okay, right? In the same way that two runners would make different sound than one runner, right? You'd expect to hear a different rhythm. So we could detect that. We don't see it. Okay. Other questions? These are great questions, you guys. Yeah, over here. So you did turn on what enzyme you have based on the AFM image before? So we only add one enzyme. So we knew that we had PK around. Yeah, and then the AFM image confirms that we have one that's attached. Okay. All right. And question in the back, Anthony. How do you actually measure the noise? Okay, I'm getting to that. Okay. Let me show you. Okay. Anthony is impatient. Okay. So you guys have exchanged songs by SoundCloud, right? So everyone, you may raise your hand if you don't know what SoundCloud is. Okay, great. This is great. You guys are totally savvy. So when you do SoundCloud, you know how like there's only this little pulse that comes with it and it tells you about the loudness of the thing? I'm going to be showing you images that are like that, data that's like that, where we're going to be watching noise. Okay. And that's shown here where this is time on the X-axis and on the Y-axis, this is the current flowing through the nanotube. So that's going to be our noise. Okay. And enzyme by itself flutters around a little bit, but for the most part, it's totally quiet. Okay. So if you don't feed the runner, you know, some oxygen or glucose, you know, some bars or whatever, the runner doesn't start running. Okay. Enzymes are like that too. Okay. Unless they get substrate, this enzyme happens to be totally quiet. Some enzymes we find kind of randomly flutter around and our technique, we will not be able to pick that up. Okay. Now, when we add ATP, we see a new blip that appears. Do you see this lower blip over here? Each one of these corresponds to the ATP bound state. So we go from up here where the enzyme is open to down here where it's bound and then back. And we can measure how long the enzyme spends in this bound state and derive a dissociation constant, a KD for this enzyme, this PKA ATP interaction. When we do that, we find that that KD corresponds to what's measured in ensemble kinetics. That tells us that actually we're seeing something very similar to what's seen in the more crowded cases. Okay. Okay. Next, we wash away the ATP and then add in a peptide that's a peptide substrate. This has the serine hydroxide that's going to be phosphorylated by the gamma phosphate of ATP. And again, we see some intermediate confirmation as the enzyme goes from open to bound. Okay. And again, do you see how there's like more blips down here? The enzyme binds to its substrate, its peptide substrate with greater affinity. It grabs on tighter. It has a lower KD. Okay. Everyone's still with me. Make sense? Okay. Now, check this out. This is the really cool one. This is now the enzyme plus ATP plus the peptide. And now we see three levels. Okay. So this is one second over here. This is now two tenths of a second. I'm zooming in. And what we see is that these three levels correspond to ATP bound and then chemtide bound, which then gives us a catalynically committed confirmation. Okay. So when the enzyme starts working, it goes between open, intermediate, and catalysis. And I'm just going to call these one, two, three. This is the waltz of pKa. Okay. So here's the enzyme waltzing going along. One, two, three. One, two, three. One, two, one, two, one, two, one, two, three. One, two, one, two, one, two, one, two, three. One, two, stuck. Three. One, two, three. One, two, three. One, two, three. So that's an enzyme in action. This is what the enzyme looks like as it goes about its business. Now what's hugely inefficient is the K off. Do you remember earlier I told you the perfect enzyme should have zero K off? In this case, we see the enzyme in real time in action being inefficient. Here it is being inefficient as it goes one, two, one, two, one, two. This is it with the K off. That catalytic inefficiency is what dooms the enzyme and makes it waste opportunities. The enzyme is trying to make up its mind. Go to product, back to substrate. Product, substrate, product, substrate. And that lack of decision is what makes this enzyme inefficient as a catalyst. There are other kinases such as kinases involved in metabolism that are far, far more efficient than this enzyme over here. If you're looking for something, could I ask you just to wait until after the class is over? Oh no, you're here for the class. You're just kind of late. Okay, no problem. Welcome. All right. So anyway, this is the enzyme in action. And what this shows us is that the enzyme fluctuates its speed enormously. Okay? And this is kind of mind boggling. And I'm just going to tell it to you. It turns out that the enzyme's speed fluctuates from second to second by a factor of 100. Okay? So this is like going out to the 73 out here, the freeway out here. And then, you know, pulling up alongside a Honda Civic. And then suddenly the Honda Civic goes from 55 miles an hour to 5,500 miles an hour. And then back down to 50 miles an hour all in the course of a second. So these enzymes are wildly changing their speeds. They're changing speeds much faster than any runner. They're changing speeds up to speeds that are wildly, that are almost inconceivable to us humans. And really, that's the stuff of life. It's essential that this enzyme is able to alter its speed in order for it to be regulated. Remember earlier, I talked about the regulation of protein kinase A. That regulation is going to control its speed. And in doing so, that turns the enzyme from being a wildly efficient catalyst to being a catalyst that's not even worthwhile, that doesn't operate on a time scale that's useful for the cell. And this is really the essence of how catalytic biology takes place. Okay? Any questions about this? Yeah? Do you see any pattern along that, the on, off, or just randomly process? Okay. This is a brilliant question. We spent a lot of time looking for patterns in our data and looking for correlation between one step or another. And we, it sure is pretty random. There is a small amount of a memory effect in the sense that the enzyme, if it hits this intermediate state, it's more likely to go down to three than it is to go back to one. Okay? And so that's actually a thermodynamic effect. That the enzyme has evolved to do K-cat in preference to K-off. All right. Let's talk about another hydra, let's talk about a different enzyme. The second enzyme I want to talk, I want to talk to you about today is lysozyme. This is an example of a hydrolase. And remember, I'm moving down, or actually I'm still at the top up here in our most common enzymes. This enzyme was discovered about 100 plus years ago and it's been intensively studied. This is the X-ray crystal structure of lysozyme. And in fact, it was the very first enzyme, X-ray crystal structure ever solved was this enzyme. The active site up here has an 8-angstrom hinge motion. And so this enzyme has kind of a Pac-Man-like motion as it hydrolyzes the glycosidic bonds of the polysaccharides found on the cell surface of bacteria cells. Okay? So here's a bacteria cell wall. And the enzyme is going to cut apart the glycosidic bond between each one of these glycan moieties found on the cell wall. Okay? So it's going to be chopping apart the polysaccharide. In doing this, this will basically burst apart the cell. In the absence of this, the enzyme is basically going to be chewing apart the bacteria. And this has the effect of killing the bacteria, right? You're breaking their cell walls. They explode, et cetera. This enzyme is found in high concentration in chicken eggs. That's the hen egg whites over here. And it's present to prevent colonization by bacteria. And you might recall, Avidin was isolated also from chicken egg whites. So biochemists have been studying what makes eggs so special as sterile vessels for a very long time. Okay. Let's take a quick look at the arrow-pushing mechanism for how this enzyme operates. In this mechanism, the enzyme goes through a covalent intermediate. Let's start over here. So I'm zooming in now to the polysaccharide region of the cell wall of the bacteria. Enzyme is going to cleave this bond that's indicated with an arrow. And this is an example of a hydrolase meaning it's going to introduce water across this bond. So the first thing that the enzyme does is torque this anacetyl-maramic acid moiety. Okay. So it's going to torque this carbohydrate from being a nice chair conformation to being a boat conformation. This is a crucial aspect for what makes enzymes such effective catalysts. This enzyme is going to be catalyzing a reaction a thousand times more efficiently than if the reaction just had to happen by itself. I actually think it's like a hundred thousand times more efficiently. In order to do this, the enzyme is going to be physically bending the substrate. And by physically bending the substrate, this helps to accelerate the reaction. So here it is pushed up into this boat conformation. Notice that the boat conformation neatly sets up an SN2 attack by this carboxylate of aspartic acid to attack backside displacement style this glycoside. Okay. This is crucial. So the glycoside gets protonated by one glutamic acid and then over here an aspartic carboxylate from a nearby aspartic acid then attacks doing a backside SN2 displacement. And this protonated glycosidic bond then is a very effective leaving group because the second arrow highlighted in red over here kicks electrons to a positively charged oxygen which is all too eager to accept those electrons. Okay. So this gives us a covalent intermediate. This is another common way that enzymes accelerate reactions. In this case we're seeing it form a covalent intermediate and then this covalent intermediate gets hydrolyzed. So half of the polysaccharide floats away. The next one comes, a water comes in, gets deprotonated by the glutamic acid, the glutamate up here. And then this hydrolyzes the ester bond between this polysaccharide and the aspartic acid. Okay. So some notable features here. We're showing, I'm showing you an example of acid-based catalysis. The enzyme is simultaneously acting as both an acid over here and as a base over here. In fact, it's even wilder than that. Check this out. It's the same functionality, this glutamic acid that acts as both the acid and the base. And to me that's just an elegant simplicity that makes enzymes so beguiling, right? If we were in the chemical laboratory trying to make molecules, you know, using glassware and such, we'd either dump in a bunch of acid or dump in a bunch of base, but you wouldn't add simultaneously both acid and base because they would neutralize each other. And enzymes have evolved to be able to simultaneously catalyze things using both acid and base catalysis. Furthermore, this enzyme has evolved to form a covalent intermediate, sometimes this is referred to as a ping-pong mechanism that eventually gives us back the hydrolyzed glycosidic bond. Okay, really, really beautiful. This is what you learn in biochemistry classes. And the problem with it is that it neglects enzyme dynamics, which are really critical. As the enzyme moves, it can then help to torque this confirmation of the ring into this boat confirmation. In the absence of this movement, it doesn't make sense really why it is that the enzyme is actually going to be torquing this substrate. Right? Because the substrate binds in the chair confirmation up here. Why should it get pushed into this other confirmation unless the enzyme is doing the pushing? And that, in fact, is what we see. Okay, so same idea. We're going to attach an enzyme to the carbon nanotube and then listen in as the enzyme works. When we do that, here's a paper from the lab from about a year ago. What we see is that, again, the enzyme by itself is relatively quiet. And then when we add the substrate, the polysaccharide, the peptidoglycan that I showed on an earlier slide, the crosslink net, there's an immediate jump upward and then there's all this noise in here. This is the enzyme chewing on the substrate and we get to listen in. It's just like SoundCloud, basically. Okay, some controls. I haven't been showing you controls, but these are everything in biology. This is substrate by itself in red, overlaid. And then this is enzyme by itself. Again, it's relatively flat and purple. And then when we have enzyme plus substrate, we see this motion where what we're seeing here is enzyme open, closed, open, closed, open, closed, open, closed, open, closed, open, closed, open, closed. This is it, you know, on a third of a second. But we get to watch the same enzyme cranking over for a long period of time. And again, what we find is that the enzyme is highly variable. It accelerates, it slows down, it speeds up, it slows down. It accesses different confirmations. In fact, it accesses at least two dramatically different speeds. And you can actually see that in the 40 seconds of data over here. Do you see how there's this dense region and then a less dense region and then a dense region? The dense region corresponds to rapid switching. The enzyme has an overdrive gear that it goes into. So it flips gears to second gear and it just starts cranking along at a much faster speed. And you can see that over here where the enzyme is going, open, closed, open, closed, open, closed, open, closed, open, closed, open, like 300 times per second. Whereas over here it's doing open, closed, open, closed, open, closed like 50 times per second. So this is dramatic. That's six times faster. And what's crazy is that the enzyme does this all day long. It switches between first gear and second gear, first gear, second gear, back and forth. And a big mystery in the field is what's up with second gear? Okay? So to address that question, I'm going to skip some stuff. To address that question, together with collaborators, we chemically synthesize a version of the polysaccharide that didn't have cross links. This is like one strand of the net that I showed you earlier. Same polysaccharide. Now we're using chemical synthesis to access a new substrate. And what we find is that the enzyme has a different type of activity. I do have to show you some more controls. Okay? I can't get away from this. They're crucial. These are mutant enzyme active sites. Do you remember earlier I showed you the carboxylates that are required for enzyme to operate? So we mutated those carboxylate residues and the enzyme no longer works and therefore it never closes. The other one of these enzyme mutations traps the covalently bound form of the substrate that I showed earlier in the ping pong mechanism. And the enzyme never can hydrolyze back off the substrate. And again, it never gets back to closed. Okay. So I get to finally tell you what the difference is between first gear and second gear. This is a day in the life of an enzyme. Here's how it spends its time. Okay? So this is an enzyme crank it along happily being fed either the linear substrate or the crosslink substrate. In the case of getting the crosslink substrate, it gets to hydrolyze things about 50% of the time. But if you feed it the linear substrate, it goes wild. It gets to hydrolyze glycosinic bonds 88% of the time. And so, and then this over here is second gear in blue. That's nonproductive rapid chatter. And over here in the crosslink, the blue is much more apparent. So in other words, the crosslink substrate, the substrate found on the surface of the bacteria cell, corresponds to second gear. So what we think is happening is the enzyme is mowing across the surface of the cell chewing contentedly. Bond after bond after bond after bond, happily hydrolyzing it all. And then it hits one of these peptide crosslinks and gets stuck. And when it gets stuck, its response is to start chattering away. It just, it flips gears and it just starts going six times faster. And what we think is happening is that it then transits along the peptide down to the parallel polysaccharide. So in the same way that DNA is a five prime directionality of three prime directionality, polysaccharides have a directionality as well. And it turns out the surface of the bacteria cell is a highway of parallel polysaccharides. So the enzyme comes along, hits across link, goes down, zooms along, hits crosslink, goes down, zooms along, down, cross, down, across. So what the enzyme is doing is zigzagging across the surface of the cell as it chews apart the surface of the bacteria. And in retrospect, this totally makes sense because again, the enzyme evolved to poke holes in bacteria. And by doing a two-dimensional rip in the surface of the bacteria, this makes the enzyme much more effective at killing its bacterial targets. Questions? Yeah. So one of the active UCs, the actual pylosys, the N1 is it transporting itself? Yeah. Well, it's a nonproductive chatter that we think is that moving along one of these peptide crosslinks. So, yeah, Anthony. So as soon as it encounters something, it can't press through, it'll switch back over. Until it finds a new glycosidic bond and then it goes to town again. Okay. Well, let's move on. I want to talk to you about other enzymes. We have lots to talk about. I want to talk to you very briefly about proteases, which cleave amide bonds. We've seen these. We've seen examples of these. In blood, there's a whole cascade of proteases that are used to respond to damaged blood vessels with a series of factor 7a, factor 10a, et cetera. Proteases where one cuts one and the next one cuts another, next one cuts another, et cetera. All the way down to the point where you get production of fibrin, which then could crosslink to form to replace to fix the damage up here. Okay. So this is an important mechanism for blood clotting. And naturally, if you're missing any one of these proteases, or one of these proteases happens to be mutated, you're in big trouble. Your blood will not clot. This happens in inbred families, such as the royal families of Europe in the turn of the century. This is the czar, Nicholas II. His wife Alexandra passed on the gene for hemophilia to their son Alexi down here. And again, this is a mutation to either factor 9 or factor 8, which are both excellent genes. These are genes that are found on the X chromosome. So they're passed along by the mother. Okay. Apatosis is also regulated by a series of proteases. Apatosis, the cell suicide mechanism that we talked about earlier in this quarter. And each protease activates the next one. So you have a protease up here called the caspase that cleaves the next caspase in line, which then cleaves this, et cetera. So this guy cleaves this guy, which cleaves this guy, et cetera. Let's take a closer look at an example of a protease. The protease I want to show you is one of my favorites. It's isolated from one of my favorite fruits, papaya. And it's perhaps appropriately called papine, because it's isolated from papaya. It's an example of a cysteine-based protease. And it's furthermore another example of a nucleophile-based enzyme mechanism. And I chose this one because cysteine, of course, is the preeminent nucleophile. As illustrated earlier today when we talked about engineering in a single cysteine on the surface of the enzyme as a way of attaching it to a specific spot on the carbon nanotube. Okay, so here's the mechanism for how this enzyme works. In practice, it's actually a fairly complicated mechanism, sorry, it's a complex mechanism, in the sense that it's a concerted mechanism. Specifically, here's the nucleophilic file functionality of the cysteine in the active site. I don't think I pointed it out here, but here's the cysteine. That's the business end of the molecule. It is active site. And in a concerted mechanism, the cysteine is simultaneously deprotonated to attack nucleophilically the amin bond. And then this amin bond carbonyl gets protonated by an acid residue that hovers above the carbonyl of the amin bond. This happens in one fell swoop from nucleophile to protonation all at once. And that is really that kind of concerted dance of catalytic efficiency is yet another example of what makes enzymes so special. The fact that everything is kind of held together at once lowers the transition state energy. Right, so now you don't necessarily have to stabilize a protonated carbonyl in an active site. Instead, you wait until electrons appear up here on this oxygen before it gets protonated. So that lowers the transition state energy for this transition state. Okay, there's other ways of depicting this as well. I prefer this one concerted mechanism. So all of these, okay, actually I'm going to skip the serine base proteases. They're similar to the cysteine base protease I showed earlier. I'm going to skip the zinc proteases as well. There's quite a few others. Like the kinases, the proteases, because they're involved in crucial processes in human physiology such as blood clotting which you would not want to happen, you know, here and there, because these are involved in such crucial processes, the enzymes themselves are tightly regulated, oftentimes regulated by some loop in a pro enzyme that has to be cleaved. Where the terminology of pro means a reaction takes place that then converts it into the active functionality of the molecule. So for example, pro drugs are precursor drugs that are then converted into the active drug by some enzymatic process. And over here, we see a pro enzyme that has a loop blocking access to the active site of the enzyme that gets cleaved and then that allows the pro enzyme piece to dissociate and turn on the enzyme. So enzymes can be very readily inhibited. You can do things like have transition state analogs. We've talked about transition state analogs before. Here is the transition state for hydrolysis of an amine bond and here is a very effective phosphor amide transition state. Notice that this also has the tetrahedral geometry of this transition state up here. And if you do that, you can actually very effectively inhibit this enzyme. Other types of inhibitors, phosphonates down here, phosphor amides over here, these KIs are the dissociation constant for binding where it's KD except it's for inhibiting, binding and inhibiting the enzyme. And again, smaller numbers equals more potent enzymes. And notice what a champion phosphor amide is with a picomolar, near picomolar inhibitor. Okay. Now, there's a million other things I could talk to you about. I'm going to pick them up next Thursday. When we come back, we'll be finishing off Chapter 6 and going on to Chapter 7. Midterm we'll cover through today's lecture.
 We're going to pick up where we left off.  Hopefully you watch the podcast lecture on Tuesday.  I want to pick up what we discussed on Tuesday, looking at protein function.  Specifically, we're going to be talking about how enzymes work.  How do enzymes catalyze key reactions in the cell and so on.  We're going to start by talking about some measures of enzyme activity.  And then we'll talk about regulation of enzymes.  And then we'll get into the mechanisms.  And then we'll close with mutagenesis and engineering.  So what we talked about on Tuesday is that proteins have a wide range of roles, structural,  binding, and catalytic roles.  In the example of structural, we saw, for example, collagen.  We saw how collagen gets organized into these complex assemblies that make it possible to  have extremely strong bones and things like that.  We also talked a little bit about Titan, a muscle protein that makes muscles capable  of being pulled and stretched without breaking, without snapping.  We also talked about binding.  The example we saw there was FKBP binding to FK506 and rapamycin.  And then finally, we're now up to the part where we can start talking about catalysis.  So protein function has at least three major roles inside the cell.  Structure, binding, and catalysis.  And when we talked about the repeat proteins, for example, we saw a great example of binding.  And I want to just very briefly, we'll take a look at that at a moment, but I just want  to emphasize something based on some questions that I got during my office hours.  We talked about how noncovalent receptor ligand interactions can be described by dissociation  constants and an on-rate and an off-rate.  We're now at the point where we're going to start talking about this, Michaelis-Menten  constant, KM, which is an analog to KD.  And something that I touched on at the very end of the lecture is enzymes work by lowering  the transition state enzyme of the reaction, energy of the reaction.  Doing this is in essence how you catalyze a reaction.  So lowering the activation energy necessary for that reaction to take place, you're increasing  the speed that the reaction takes place by doing this.  And enzymes do this by binding to the transition state and stabilizing it.  By having counter ions that stabilize charge functionalities in the transition state, the  enzyme can lower the transition state energy.  And this is surprisingly effective.  We're going to be seeing some examples of that today.  So we're going to be talking today also about how this catalysis by enzymes is coupled to  the motion by the enzyme.  And I'll show you some examples of this.  And what we're going to see is that enzymes force the substrate, the starting material,  into confirmations that favor formation of the product.  And by doing that, that accelerates these reactions.  Okay, and very briefly, I just want to touch on one point about the repeat proteins.  Something I didn't mention but probably should have when I talked about the repeat proteins.  This is an example of a repeat protein.  This is an anchorin repeat.  Notice that it has a series of helix, turn, helix, turn, helix, turn, helix, turn, helix,  turn, helix, turn, helix.  So those repeats, each one of these loops and then helix, loop and then helix.  And notice that they're all stacked on top of each other.  Each one of those is one example of a repeat.  And this anchorin repeat has a whole bunch of these lined up.  Okay, that's where it gets the name repeat protein.  Make sense?  And then similarly with the leucine rich repeats, in this case, it's a helix, strand, loop,  helix, strand, loop, helix, strand, loop, et cetera.  Okay, but it's a series of repeating motif, repeating structural motif.  In fact, these are actually individual domains.  They fold modestly well on their own and they can be shuffled around to a limited extent.  Okay, any questions about the topic of protein structure?  And for that matter, any questions about anything that was covered on Tuesday?  In Tuesday's lecture.  All right, well I think in that case then we're ready to move on.  We're going to talk next about catalysis.  Today's discussion is going to be pretty high level.  I'm going to be telling you stuff that's actually not in the book.  And in fact, actually it's only found really in the frontiers of the literature  in chemical biology.  So don't hesitate to interrupt if at any point I start to lose you.  Okay, it's better if you interrupt me early on than if I go further down the road  and then you're totally lost.  Okay, because the truth is this lecture will be the only time that you're going  to be able to find this material I'll be discussing.  And I think it's very, very important.  It's actually the very frontiers of chemical biology.  Okay, so I want to talk to you today about catalysis.  And last time I showed you that non-covalent binding consists of ligands hopping  on to some binding site in a receptor.  And we describe this non-covalent interaction using an equilibrium constant,  a special equilibrium constant called a dissociation constant  that quantifies the ratio between unbound up here  to bound receptor ligand interactions.  Okay, so this is pretty straightforward stuff.  The only difference with catalysis is that we're going  to have this similar receptor ligand interaction.  But upon binding to the ligand, the enzyme in this case instead of the receptor,  and for that case instead of the receptor, the ligand is going to be transformed,  converted into some new product.  So it's a similar sort of process.  So if we understand non-covalent interactions,  then we can also understand catalysis by enzymes.  Okay, I'm skipping ahead, skipping, skipping.  We talked about this already.  All right.  This is, I believe this is where we left off, right?  Is this right?  Okay, so where we left off again is this idea that receptor ligand interactions are governed  by some binding.  In the case of enzymes, the substrate binds and then some catalysis takes place  to convert the substrate into a product.  S stands for substrate or starting material, and that gives us P, which is the product,  and then the product has to dissociate.  I think I've talked about this, right?  This was covered.  Right? I did cover this.  Okay, good.  Right, okay, so I think I'm actually over here.  Okay, good.  So here's a typical reaction scheme I already talked about on the previous thing.  So the formation of this enzyme substrate product, this mechalosmentin complex,  this is sometimes called the mechalosmentin complex, this complex here  of the activated enzyme bound to the substrate in a way to catalyze the reaction has an equilibrium  constant, and in the same way that the KD was proportional to the rate constants  for the dissociation, the K off to K on, in the same way the mechalosmentin equilibrium  constant is equal to the sum of the off rate and the K cat.  So that's like getting to the ES and either going backwards or forwards.  That's the rates going in either direction for destruction of the complex versus forming the  complex, meaning the rate constant for K on.  This KM resembles the KD for non-covalent binding interactions,  and so it's useful for us.  The strength of the KM tells us something about how avidly the enzyme is going to grab  onto the substrate and how quickly it's going to form this ES complex.  So that, this KM is actually a useful number.  It tells us something about the conditions inside the cell  because the enzyme has to evolve up to a sufficient affinity for its substrate  so it can grab onto the substrate inside the cell.  If the affinity is too low, then the enzyme substrate complex will never form  and the enzyme will never catalyze a reaction.  On the other hand, if it evolves to the point where it's super-duper high,  maybe that's not so useful for the cell  because maybe the enzyme then will be a little too active.  So these evolve up to the maximum ability that's necessary for the cell.  That's required for the conditions found inside the cell.  And oftentimes, for example, we can engineer enzymes to have very different KMs simply  by tinkering with their active sites.  And I'll talk about that more in a moment when we talk about protein engineering.  Okay, so if we look at a dose response diagram, this is similar  to the dose response diagrams that I showed on a previous slide on Tuesday.  Instead, on the y-axis, we have our initial reaction velocity and the concentration  of the substrate, the point of inflection here is going to be roughly the KM,  this equilibrium constant up here.  For that matter, this point of inflection also tells us where 50%  of the maximum rate of the enzyme is going to be.  At the very highest concentrations of substrate, the enzyme is going to be running flat out.  Okay, so that's like as fast as the enzyme can possibly go.  It would be the equivalent of giving the sprinter maximum oxygen, maximum glucose.  Everything he or she needs to do to run as fast as possible.  So that's up here under maximum velocity conditions.  Notice that this asymptotically approaches this V max value way up here.  Okay, and so anything where it's an excess concentration of substrate  that's called V max conditions and typically enzyme reactions that are run  in the laboratory are run under those conditions.  We always have an extreme excess of substrate typically.  Okay, let's take a look at some KMs.  They range widely.  There's a wide range of possible KMs for enzymes.  And here are some numbers over here.  Now, like the KD, a lower KM value means tighter binding.  Totally analogous to the dissociation constant.  In fact, it has a very similar connotation.  So what this tells us, for example, is that this enzyme here,  cytochrome P450, binds benzopyrene with theory high affinity.  This should come as no surprise to us.  Benzopyrene is this big, flat hydrophobic molecule.  And hydrophobic molecules in general aren't so soluble.  So the cytochrome P450 in your liver is going to be grabbing  on to the benzopyrene that you inhaled on your way to over here  when you got behind that stupid shuttle bus  that was, you know, dinking along at too slow a speed.  Right? So you get behind the exhaust pipe of that shuttle bus  and you start inhaling unburnt benzopyrene.  So this cytochrome P450 in your liver is right now, as we're speaking,  grabbing on with great affinity for these benzopyrates.  On the other hand, there are some enzymes that don't have to grab  on all that well to their substrates like aconitase.  This is a key enzyme in the metabolism of glucose.  And its substrate, citrate, is found at high enough concentrations  that the enzyme doesn't really have to evolve  to a very high affinity.  So this gives us sort of a crude measurement  of what the concentration of the substrate is in the cell.  Right? So what we know is that there's probably not a lot  of benzopyrene present, but there's probably tons  of citrate present, hence the need for lower affinity.  Now, let's also take a look at some K-cats.  So this is the equal, this is the rate constant  for the decomposition of the macalus menten complex,  the ES complex that is now being broken down  to form enzyme product.  Okay? Make sense?  Okay. So in this case, again, there's a wide range of K-cats.  And this tells us something about how hard the reaction is  to catalyze.  Harder reactions in general have lower K-cats.  But it can also tell us something about the evolution of the enzyme.  Enzymes in general evolve up to the required function  and really don't go past that.  Okay? There's really no evolutionary drive.  There's no selection mechanism that drives the enzymes  to be perfect unless they need to be perfect  for some particularly crucial function for the cell.  So here's one example of a really crucial function for the cell.  The enzyme catalyze breaks down hydrogen peroxide  into oxygen and water.  This is a crucial reaction.  Hydrogen peroxide creates a substantial burden on cells.  This is a strong oxidant.  And oxidants run around and wreck havoc on cellular machinery.  And so for this reason, cells have evolved pretty sophisticated  mechanisms to very quickly break down such oxidation products.  And catalyze has a K-cat of 100 million.  So this is a really, really fast catalytic reaction  that takes place.  And then a slower reaction would be a protease.  Protease is of course hydrolyzed amide bonds.  I believe we've seen these before.  And their K-cats are much lower likely because this reaction is a little  more challenging and a little less favorable thermodynamically.  And also for that matter, it's not as critical perhaps for the cell.  Okay. Questions so far?  Good. Okay.  So these are the numbers that are going to underlie our discussion  as we start talking about the properties of enzymes.  Okay. These are the same numbers that you learned about in like Bio 99  or 98, whatever biochemistry class you take here at UC Irvine  or elsewhere.  These numbers are kind of the vocabulary  that my biochemistry friends talk about when they talk about enzymes.  Okay. Now the truth is as a chemical biologist,  I don't get too worked up about these numbers.  I'm more interested in understanding the atoms and bonds basis  for how the enzymes work.  And so I guess the best place to start would be let's start  with the perfect enzyme.  What would be the enzyme that really can crank,  that could maximize its ability to turn over a reaction?  And then we'll look at some specifics at the level of atoms  and bonds.  So the very perfect enzyme, you might imagine,  every time it forms this Michaelis Menten complex,  the ES complex, then it goes immediately to Kcat.  So it forms and then boom, it's over to the Kcat.  And it just immediately gets converted,  converts the substrate to the product that happens instantly.  On the other hand, the perfect enzyme is not going  to have any off rate over here.  This off rate represents lost opportunities.  This is the sea, the wasteland, coulda, shoulda, maybe shoulda.  Okay, right?  This is the chance that the enzyme missed.  So instead of going to product, the enzyme goes backwards.  And so this off rate over here is miserable and inefficient  for an enzyme.  So the perfect enzyme is not going to have an off rate.  And so the perfect enzyme, you can basically imagine Koff being 0.  And if we have that, then we can imagine rearranging our KM  equation shown a couple of slides ago,  such that Kon equals the ratio of Kcat to Km.  And again, notice that these little k's are indicating rate  constant and the big Km is for equilibrium constant.  So the very best enzyme will have an on rate  that's diffusion controlled.  In other words, it's limited by the amount of time  that the substrate, Brownian motion style,  eventually bounces its way to the active site.  That should be the slowest step for an enzyme that's perfect.  And we've talked about this before, but that rate  of diffusion has a speed limit of 10  to the 9th per molar per second.  It can't go any faster than that.  That's a physical law.  It's like the speed of light.  You cannot exceed that.  Just because it takes a little while to bounce around through all  that water and other stuff that's present in the cell.  Okay, make sense?  We'll take a look at a moment at an example of an enzyme that's far  from perfect and we'll start to understand what its sources  of imperfection are.  So before we do, let me just give you a little table  that I really like that shows us  and helps us organize enzymes.  This shows us the rankings of enzymes in your proteome.  Okay, so this is a listing from most common to least common.  It's like a greatest hits of the seven categories of enzymes  that are found in the human proteome.  The most common enzymes by far are the hydrolysis.  These are the enzymes that introduce water as a way  of breaking a bond and we're going to see a couple  of examples of this.  We'll see examples of glycosylases and proteases today.  We've already seen examples of nucleases.  That was stuff like RNAase, right?  Remember when we talked about RNAase and it was inhibited  by Dipsy?  This is a similar sort of thing.  Can someone help this guy out?  Thank you.  Okay, transferases, next most common, second position.  These are examples of enzymes that transfer functionality  from one spot to another and we're going to look in detail  at an example of a protein kinase today  and then later in the class we'll look  at a glycosyl transferase.  Oxido reduptases, this is like the enzyme cytochrome P450  that takes the benzopyrines, introduces an epoxide  and oxidizes the benzopyrines.  You all remember this, right?  I showed you the benzopyrine a couple of slides ago but earlier  in this quarter when I was talking about cigarette smoking,  I showed you how the benzopyrine that looks  like this rather innocuous flat structure gets converted  into an epoxide and then slips into the pi stack of your DNA  and alkylates the DNA.  And so these are actually very common enzymes,  these oxido reductases because they're important  for removing toxins.  Dehydrogenases are another one that's very common  and perhaps we'll get a chance to see this one today.  And then finally we get down to the ligases.  These are enzymes that spot weld together to functional groups  such as attaching ubiquitin or DNA to something.  Dysomerases are used to convert substrate  into some related isomeric product.  These are things like epimerases.  The synthetases we've seen before,  we talked about aminoacyl tRNA synthetase.  This was that gargantuan complex that read out the anti-codon  and the various modifications of the tRNA to make sure  that the correct amino acid was being attached to the tRNA  during aminoacyl tRNA synthesis.  Okay, so and then the final one, the liases are doing things  like decarboxylation.  These are actually breaking carbon-carbon bonds  in dramatic fashion.  So these are aldolases that are doing aldol reaction,  et cetera.  They're either breaking or making carbon-carbon bonds.  So fairly, I feel like we've seen many examples  of these different enzymes this quarter.  So now I can go through and just talk about the ones  that are really important that we haven't seen yet, okay,  such as the kinases over here.  And I believe that's where I'm going to start.  Yes, in fact it is.  So it turns out that kinases have a common fold that consists  of a lower domain down here and then a larger lobe up here.  The active site is indicated  where these Van der Waals spheres are.  This is ATP.  So kinases take ATP and transfer the gamma phosphate  of ATP to some sort of hydroxide recipient.  Okay, that's generically what they're doing.  When we talk about the gamma phosphate, ATP,  adenosine triphosphate, ATP has three phosphate groups called  alpha, beta, and gamma.  The third one in the row is called gamma.  And so that's the phosphate group  that the kinases are going to transfer.  So again, notice that these have a conserved dual lobe structure  even though these have widely disparate activities.  This is everything from a receptor tyrosine kinase  over here to protein kinase over here.  These do have very different activities.  They phosphorylate different targets.  And yet on the other hand, they all evolved  to have very similar structures.  Now, this class of enzymes, like all enzymes,  can be inhibited by substrates, pseudo substrates  that mimic the real substrate.  So here's the structure of ATP, but in place of one  of the oxygens of ATP highlighted in blue, we have a nitrogen.  And this phosphoramide inhibits the kinase.  Okay, so if you feed this phosphoramide to a kinase,  to any of the kinases I showed on the previous slide,  it's going to be game over for them.  They're not going to be able to work because they're going  to bind to this ATP analog.  They're going to put it in a sloppy old beer hug,  but this gamma phosphate is missing the oxygen.  And missing the oxygen is the same as saying it's totally inert.  And so this is going to basically be locked in the active site,  in the brace of the active site,  yet unable to transfer the phosphate group.  And so the net effect is to shut down the enzyme.  And this is a very effective way of killing enzymes.  You basically know something about the mechanism.  You make a tiny little modification of the substrate,  and boom, it's game over for the enzyme.  Okay, so you could do this also with sulfur or nitrogen.  So I have shown you the nitrogen, but you could do that as well.  And again, it sticks in the active site and inhibits the enzyme.  This approach also works if you mimic the product.  And a large number of enzyme inhibitors pursue one  of those two approaches.  Either mimicking the substrate, as shown here,  or mimicking the product.  Either approach works great.  So let's zoom in.  I showed you the structure, the bilobe structure of kinase.  Let's zoom in and take a look at its active site.  In the active site, there are, here's the structure of ATP.  There are a series of conserved magnesium ions,  these balls over here, that are bound  to the phosphate groups of the ATP.  The numbers here indicate an angstrom the distances.  Okay, and these numbers are pretty low.  Right, if you recall that a carbon-carbon bond is somewhere  on the order of like 1.5 angstroms or so.  These are pretty close in numbers, right?  These, this magnesium is getting awfully close  to this oxygen over here.  These are cozy, cozy molecules and atoms.  They like being this close.  They like being this close because they have complementary  charges, right?  The magnesium has a plus 2 charge.  The oxygens of these phosphate groups have negative charges.  So they're attracted by salt bridges or coulombs interactions  that we saw earlier in the quarter.  So over here, there is the other substrate  for this enzyme reaction that has a hydroxyl.  I'm showing it to you with the hydroxyl deprotonated.  And after a phosphoryl transfer, this oxygen  of the substrate has now picked up a phosphate group.  And notice that the magnesiums here are helping  to stabilize that phosphate group, right?  That they're lowering the energy of binding by forming that same  sort of coulombic salt bridge that we saw earlier.  Okay, now if we zoom in and take a look at the arrow pushing  of, arrow pushing mechanism for this enzyme active site,  what we find is that and not depicted  on this previous slide, somewhere out here,  there's a carboxylate side chain from aspartic acid.  The carboxylate of this aspartic acid deprotonates the proton  of the hydroxy of a serine for the substrate.  And that sets us up with an alkoxide.  Alkoxide being a superb nucleophile, it's negatively charged,  can attack the gamma phosphate of ATP.  And again, the magnesiums get in on the action.  They're over here participating fully  and stabilizing this negative charge  of the phosphate group.  That's crucial, right?  You can imagine this reaction not going in the absence  of those magnesiums, right?  Because one negative charge is not going to want  to approach a negatively charged phosphate group, right?  The negatively charged alkoxide over here is going  to be stymied in its attack.  It's going to get repelled by this phosphate group.  So the magnesiums are shielding the phosphate group  and protecting it and preventing it from getting,  from looking like a negative charge.  And so that tees up this reaction very deeply.  And then finally there's a collapse  of this trigonal bipyramidal intermediate.  And just very briefly, the structure  around this phosphate looks like a trigonal bipyramid.  Not so, anyway, that's interesting.  And then there's collapse  of this trigonal bipyramidal intermediate  to give us our final product.  Okay, so to summarize, the most important aspect  of this is the notion that the magnesium ions are playing  several roles to make this reaction possible.  First, they're coordinating and stabilizing the transferred  phosphate group as a Lewis acid.  Okay, so that helps accelerate the reaction  as a Lewis acid.  Turns out that Kine's activity  in the cell is very tightly regulated.  And the reasons for this are perhaps not clear  if you don't know much about signal transduction.  And I'm just going to very briefly cover it today.  Then in a future lecture, we'll learn quite a bit more  about it.  In the cell, there's a series of pathways  that transfer information.  And these pathways are controlled by transfer of Kine,  transfer of phosphate groups to key residues and proteins.  So Kine's play a really key role  in kicking off various processes in the cell.  So there's cascades of Kine's  where one Kine's phosphorylates the next Kine's  which phosphorylates the next Kine's and so on and so forth.  Turns out that this process is very tightly regulated  because you don't want your cells going wild.  You don't want them to be doing uncontrolled cell division,  for example.  And so for this reason, the cell very tightly regulates Kine's  activity.  And I want to show you a couple of vignettes  about this tight regulation because it's crucial  to our understanding of how Kine's work.  Okay, so here's one example.  This is an example from the enzyme protein Kine's A,  cyclic AMP regulated Kine's.  And the way this works is there's actually a regulatory subunit  shown here in blue that binds to the Kine's  and actually has an inhibitory loop  that blocks access to the active site.  Okay, so does everyone see how this dark blue thing is?  It's binding here and then it has like this long finger  that fits into the active site and blocks the Kine's  from binding to any substrates.  This shuts down the Kine's and the ability  to shut off the Kine's is crucially important.  Okay, if you don't have this, the Kine's will be running  around rampant wrecking havoc, turning on stuff,  shutting off stuff, causing death and destruction  and general mayhem.  And I do mean death and destruction.  Okay, these Kine's are that important.  Now, when levels of a reporter molecule called cyclic AMP reaches  certain concentration, this cyclic AMP binds  to the regulatory subunit and causes the regulatory subunit  to dissociate from the catalytic subunit approaching Kine's A.  So these two molecules get forced apart  as the blue one flips into a new confirmation.  Upon binding to cyclic AMP, the thing changes its shape  and it no longer has affinity for protein Kine's A. This is good,  this is your protein Kine's A. It frees it up to go off  and do the mission that it's wanted to do for its entire life,  which is to run around the cell  and phosphorylate anything that moves.  Nearly anything that moves.  It actually has a little bit of specificity,  but for the most part, protein Kine's A likes to phosphorylate lots  of different binding partners.  Okay, this is a pretty promiscuous molecule.  Now, here's the thing.  Another way of regulating enzymes is to phosphorylate them.  Okay, so this is one way  where you have some regulatory protein that binds.  A second way is to phosphorylate residues  that are near the active site.  Okay, so for example, this non-hydrolyzable analog of ATP,  which has the nitrogen in place of oxygen.  This is the molecule I showed on a previous slide.  This tells us where the active site is.  But over here are two residues that can be phosphorylated  to flip on this map Kine's, this P38 gamma map Kine's.  And so one of these is a tyrosine and the other one is a serine.  And so this Kine's waits around until it gets to the,  until it gets phosphorylated and at that point it goes into gear.  Okay, so this is like an on-off switch for the Kine's.  In the absence of this,  the enzyme doesn't have the right confirmation.  It doesn't have the right confirmation.  It can't be a Kine's.  Okay, so the phosphorylation of the Kine's puts it in gear,  turns it on, and sets it going.  Does this make sense?  Any questions about what you've seen so far?  Okay, that's the basics.  I want to talk to you about the really neat stuff,  the latest results in thinking about how Kine's is work  and thinking about their motions.  And again, this is kind of an abrupt departure  from sort of standard material as presented  in biochemistry classes.  And it really represents the frontier in chemical biology.  Many of the next experiments I'll be talking about were done,  actually, with Miriam.  She's one of the leaders in this area.  Okay, so the thing is I want to talk to you  about how enzymes work at a mechanistic level  and how they actually work dynamically.  How do they move when they do these reactions?  So I should tell you that enzymes have great motions  associated with their activities.  This is unlike the case of conventional catalysis  by organometallic complexes that you learned  about back in Chem 51, okay?  Or if you learned about in Chem 125.  In those cases, the organometallic catalyst binds  and perhaps it plays some Lewis acidic role,  but it's certainly, we don't think about its motion.  Okay, we don't think about it having some, you know,  movement associated with it, some dynamics.  Enzymes, it turns out, for the most part,  almost all have very wild and very quick motions associated  with them and a frontier in chemical biology is  to understand how those motions impact catalysis.  How do those motions allow enzymes to be effective catalysts?  And so it turns out that if you get a big, you know,  round bottom flask full of enzymes, you'll never be able  to see those individual motions.  And the reason is they tend to get blurred out, okay?  So if we look at a large number  of molecules, we'll never see the individuals in motion  because all of the enzymes in that flask are going to be running  along at different speeds and everything gets blurred out.  Okay, so instead, in order to see individual motions,  we have to look at single molecules.  And to understand this a little bit better,  let me offer an analogy.  Let's imagine that I convinced, you know,  the Orange County Marathon folks to reroute the marathon.  So instead of being on Sunday morning,  instead I convinced them to run it here Thursday at 10, 10 a.m.  And I convinced them to start at that end of the classroom  and send all the runners through the classroom  from that door to this door.  Okay, so now everyone's running  through here all 10,000 runners.  You can imagine what you're going to see is just a blur  of pumping arms and legs, right?  Everyone's going to be trying to get in and out  of this classroom as quickly as possible.  It's going to be total pandemonium.  That's the situation when we look at an ensemble of enzymes.  It's total pandemonium.  It's a blur of arms and legs.  We don't see anything.  Everything gets averaged out.  And by see, I mean using tools like spectroscopy,  using tools that you're familiar with from your other classes.  So over the last 15 years, there's been a revolution  in this area of chemical biology or biophysics  where scientists have started to look  at individual molecules in isolation from all  of their other friends and neighbors.  Okay, so now instead of having the entire marathon coming  through the classroom, let's imagine that I convinced each  runner to come running through one at a time.  So they're going to start over there  and then come running through here.  What you will see because each runner is isolated  from all the other runners is you'll be able  to see their arms and legs moving, right?  Because now there's no blurring out effect, right?  And furthermore, if you look closely,  you'll be able to see some runners moving faster than others.  Maybe one runner has a different stride than her neighbor,  right? Because she comes running through and I don't know,  maybe she extends her leg a little longer  than the runner behind her.  Okay, but if we convince them to be isolated from each other,  then we can really start to get information about how they move.  And that's the situation we find ourselves  in when we start looking at enzymes.  And so this area of single molecules allows us to look  at conformational steps, at intermediates, and the kinetics  and dynamics that underline enzyme function.  And this again is a major frontier  and it's a really exciting area to be involved in research.  Okay, so everyone with me so far.  Everyone understands this idea of looking  at single molecules, right?  Okay, good.  I want to talk to you next about how we're going  to observe our single molecules.  There are a number of different fluorescence techniques  for looking at single molecules.  Patch clamping you may have heard of is a 40-year-old proven  technology that works really well for looking  at individual receptors.  That works fine.  In the last five years or so, groups here at UC Irvine have been  at the forefront for inventing sort of a tiny little microphone  that allows us to listen in to enzymes as they run.  And it has some advantages over those other techniques.  And so that's what I want to talk to you about today.  Okay, so this is a collaboration between my laboratory  and Phil Collins in the Department of Physics here  at UC Irvine.  And he's pioneered ways of building circuits that are based  on carbon nanotubes.  And this is an example of a carbon nanotube.  This is basically a carbon graphite graphene layer  that's folded up into a cylinder.  It looks sort of like chicken wire.  These wires though are amazingly conductive.  Carbon nanotubes are really remarkable material.  They have remarkable mechanical properties.  They have remarkable properties for conducting electricity  and for conducting heat.  In terms of conducting electricity, all of the electricity is  going to be flowing through the outside of the wire,  through these bonds out here.  And the electricity is not flowing through the middle  of the wire.  So this is unlike, for example, the copper wires that are used  in wiring the walls, okay,  wiring the electrical outlet over there on the walls.  And this property makes the outside of the wire superbly  sensitive to tiny little perturbations on its surface.  And that's what we're going to do.  So here's the way we do this.  Students in the Collins laboratory and my laboratory start  with silicon wafers that are about this big.  We go to the engineering building across campus.  The students put on bunny suits.  And we build using photolithography circuits  that look like this.  There are these contact pads to which we attach wires.  And then down here, you get down to these interdigitated  electrodes that do not touch each other.  So this is an open circuit.  But somewhere out here, we sprinkle an iron militant  of catalyst that catalyzes growth of one  of these carbon nanotubes, a single walled carbon nanotube,  across the wires to complete the circuit.  And I false colored it in red over here.  Okay. So now what we do next is we turn this wire  into the world's tiniest microphone.  We turn it into a device called a field effects transistor.  It's not so important how that works.  What matters is it's more or less the same  as the microphone found in my cell phone.  Okay. Same principle.  And next, we're going to glue individual proteins directly  onto the microphone and listen as they flap around.  Okay. So runners, if we had runners running through here,  you'd expect to hear the pounding of their feet, right?  And you'd expect to be able to interpret the noise  of their feet to tell us something about their stride,  whether or not they're accelerating,  whether or not they're slowing down, et cetera,  whether or not they have funny heel strike, et cetera.  Right? Makes sense?  So we're going to do the same thing but with proteins.  Now, I know that you're probably thinking proteins don't have noise.  I have proteins all over my body  and I'm not hearing anything right now.  And the truth is the noise is very, very tiny.  It is so tiny that it's very hard to hear.  But moving charges do make noise.  And I'll give you one example of this.  If you're at the beach with a bonfire down here  at Little Corona Beach State Park  and you have this big bonfire going, you know how the wind kind  of whips the flames and the flames make this kind  of neat, whooshing noise?  That sound of the flames moving is due to plasma  in the flames that's charged ions in the flames  that are moving around.  So charged functionalities make noise as they get pushed around.  And in fact, actually there's a speaker, a loud speaker.  These are really expensive stereo speakers.  On the order of like $20,000 a pair.  They better sound good at that price.  But it's actually based upon having a plasma that's moved  around by a little magnetic coil.  Okay? So you can actually hear charged ions moving around.  And that's what we're going to do when we glue in the protein.  So let me show you what it looks like when we have the protein  glued in.  This is the schematic diagram over here.  We have the carbon nanotube.  Here's the protein glued in.  This protein is streptavidin.  Familiar, right?  Everybody in this classroom.  And we have streptavidin conjugated to a tiny little dot  of gold and that's shown here.  Okay? So the little dots here, that's the gold attached  to streptavidin.  And then the horizontal lines are the wires,  the carbon nanotubes.  And the vertical are the electrodes over here.  And you can see we're getting one, one, one, one, one attachment.  Okay? So the breakthrough that Phil and I came up with,  with our coworkers, our friends, the graduate students,  was that we developed a way of attaching one  and only one attachment each time to the carbon nanotube.  This means then that we're isolating the enzymes away  from all their buddies, which means then we can start looking  at confirmations and intermediates.  Okay? This is all a long introduction.  We're going to get back to the kinases in a moment.  Before I do, let me just set the stage.  Here's the experiment again.  We have the electrodes.  We have the carbon nanotube.  It turns out that, of course, you can't run your cell phone  in water, so you can't run one  of these tiny microphones in water.  Okay? I think I did this experiment last week.  I dropped my phone in a bucket of water.  Actually, it was my cat's water bowl.  And I pulled it out quickly enough,  but it definitely did cause some damage.  So electronics and water don't mix.  I don't think it surprises anyone in the classroom.  And so for what we have to do is we have,  but all biology, of course, takes place in water.  So this creates a dichotomy.  And to solve this, what we do is we cover up all the electronics  with a layer of polymethylbethacrylate.  This is shown here in gray.  And then we blast a little tiny window using something called  an electron beam that just opens up a little tiny region  of the carbon nanotube.  And that's where we're going to do the experiment.  Now all the images I've been showing you up till now are  electron micrographs using electron microscopy.  We're now going to get really small as we start imaging individual  molecules of proteins.  This is so small that you can't really see them very readily  using electron microscopy, except if you use that trick  that I showed on a previous slide where you coat things  with gold, that was just streped out of the thing.  So now to see these things, we now are going to have  to use atomic force microscopy, where we're now getting down  to really tiny resolutions of one nanometer or so.  So here's what it looks like.  This is one enzyme attached to the carbon nanotube.  And this is an AFM image, atomic force microscopy image,  just showing the windowed region,  just showing the carbon nanotube that's exposed.  This little blob right here is actually the enzyme attached.  It has the right dimensions for that one enzyme.  And now we turn on the microphone  and it's lights camera action.  Okay, at that point then we're ready to listen in.  Okay, one more image.  This is before and then that's after.  And where it's circled, you can see very clearly the enzyme  attached.  Okay, everyone's still with me.  Questions so far?  All right, yes?  What are the other?  Oh, thanks for, yeah, thanks for these other blobs  that are kind of like, those are other little enzymes  that we can't get rid of.  It's actually enormously hard to do these sort of images.  It just turns out the proteins are kind of sticky.  There might even be some salt crystals somewhere out here.  So there's always some garbage-y stuff  that we've been totally unable to get rid of despite a lot  of work.  It took a lot of work to even get images that are this clear.  So.  Could it be different when an enzyme gets separate from others  the way they add the react and the ray in some?  Okay, that's a good question.  Okay, that's a really good question actually.  Thanks for not asking that.  Thanks for not being on the study section that asked that.  Okay, so the question is, would an enzyme in isolation  from itself behave differently than an enzyme that's next  to its neighbors, right?  In the same way that a crowded field of runners is going  to run differently than if a solo runner.  We don't know.  I would like to think it's going to run the same.  But it is a legitimate caveat and I thank you for that.  I will have to think about that some more.  Thank you.  So why do we study that?  Why are we not in fact, you know, well-increased  in that?  Yeah, so enzymes frequently aren't really, so it's true.  Enzymes are really crowded conditions inside the cell.  But it's not like there's like a thousand enzymes  that are all doing the same thing crowded together.  It's more like there's a couple enzymes that are kind  of jammed in with hundreds of other molecules inside the cell.  You know, so they're not all doing the same thing.  So we can recreate that sort of thing.  We can recreate the crowded conditions inside the cell  and compare.  We haven't gone into the experiment, but I'd love to do it.  So, okay?  Okay, let's get back to kinases.  So again, this is protein kinase A. It still has the two lobes  that I showed earlier.  Here's the big lobe down here and here's the smaller lobe up here.  And somewhere close to the, this upper lobe, Miriam,  together with Issa Moody, a former graduate student  in the laboratory, engineered a single cysteine.  The cysteine, of course, has a sulfur functionality,  a thiol functionality, and that allows us  to attach site specifically the enzyme to the nanotube,  to this microphone down here, through a pyrene.  Okay, so pyrene is making yet another cameo  in today's lecture.  And as you might expect, the pyrene is going to py-py stack  onto the carbon nanotube.  Okay, because it's so hydrophobic, it's looking  for a nanotube to stick onto.  And it turns out the enzyme is very firmly held in place.  This is like a very special kind of molecular glue  that sticks these two molecules together.  But notice that it's being held  in a non-covalent interaction.  And in practice, this thing is held in place for 10 or 12 hours.  We don't see it coming off.  It's really stuck in there very firmly.  Okay, and again, here's another AFM image.  And that little blob attached  to the carbon nanotube is our enzyme.  Okay, yeah, question over here.  Sergio.  How do we ensure that we have just one enzyme attached  and not two in the liquid?  Yeah, so we do AFM, this technique  of atomic force microscopy,  before we get started with the experiment.  Just to make sure that we have one attachment.  And we use this special technique called in liquid AFM.  If we see two attached, we wash it away,  and then start over.  If we see zero attached, we start over with attachment.  Okay, yeah, question over here.  So when you have one and you use AFM,  I'm sure you have one.  Yeah.  You know, like for example, a second one doesn't attach  afterwards when you start listening.  Okay, so afterwards, we don't add any more enzyme.  So we have like purified buffer that has no enzyme around.  So, and I know what you're thinking.  You're wondering, well, what about this blob over here?  What if it decides to get up and wander over here?  It turns out these blobs are pretty firmly stuck  down on the surface.  And another thing is if another one attached,  we'd hear that one running alongside it.  Okay, right?  In the same way that two runners would make different sound  than one runner, right?  You'd expect to hear a different rhythm.  So we could detect that.  We don't see it.  Okay. Other questions?  These are great questions, you guys.  Yeah, over here.  So you did turn on what enzyme you have based  on the AFM image before?  So we only add one enzyme.  So we knew that we had PK around.  Yeah, and then the AFM image confirms  that we have one that's attached.  Okay. All right.  And question in the back, Anthony.  How do you actually measure the noise?  Okay, I'm getting to that.  Okay. Let me show you.  Okay. Anthony is impatient.  Okay. So you guys have exchanged songs by SoundCloud, right?  So everyone, you may raise your hand  if you don't know what SoundCloud is.  Okay, great.  This is great.  You guys are totally savvy.  So when you do SoundCloud, you know how like there's only this  little pulse that comes with it and it tells you  about the loudness of the thing?  I'm going to be showing you images that are like that,  data that's like that,  where we're going to be watching noise.  Okay. And that's shown here where this is time on the X-axis  and on the Y-axis, this is the current flowing  through the nanotube.  So that's going to be our noise.  Okay. And enzyme by itself flutters  around a little bit, but for the most part,  it's totally quiet.  Okay. So if you don't feed the runner, you know,  some oxygen or glucose, you know, some bars or whatever,  the runner doesn't start running.  Okay. Enzymes are like that too.  Okay. Unless they get substrate,  this enzyme happens to be totally quiet.  Some enzymes we find kind of randomly flutter around  and our technique, we will not be able to pick that up.  Okay. Now, when we add ATP, we see a new blip that appears.  Do you see this lower blip over here?  Each one of these corresponds to the ATP bound state.  So we go from up here where the enzyme is open to down here  where it's bound and then back.  And we can measure how long the enzyme spends  in this bound state and derive a dissociation constant,  a KD for this enzyme, this PKA ATP interaction.  When we do that, we find that that KD corresponds  to what's measured in ensemble kinetics.  That tells us that actually we're seeing something very similar  to what's seen in the more crowded cases.  Okay. Okay.  Next, we wash away the ATP and then add in a peptide  that's a peptide substrate.  This has the serine hydroxide that's going to be phosphorylated  by the gamma phosphate of ATP.  And again, we see some intermediate confirmation  as the enzyme goes from open to bound.  Okay. And again, do you see how there's like more blips  down here?  The enzyme binds to its substrate, its peptide substrate  with greater affinity.  It grabs on tighter.  It has a lower KD. Okay.  Everyone's still with me.  Make sense?  Okay. Now, check this out.  This is the really cool one.  This is now the enzyme plus ATP plus the peptide.  And now we see three levels.  Okay. So this is one second over here.  This is now two tenths of a second.  I'm zooming in.  And what we see is that these three levels correspond  to ATP bound and then chemtide bound,  which then gives us a catalynically committed  confirmation.  Okay. So when the enzyme starts working, it goes  between open, intermediate, and catalysis.  And I'm just going to call these one, two, three.  This is the waltz of pKa.  Okay. So here's the enzyme waltzing going along.  One, two, three.  One, two, three.  One, two, one, two, one, two, one, two, three.  One, two, one, two, one, two, one, two, three.  One, two, stuck.  Three. One, two, three.  One, two, three.  One, two, three.  So that's an enzyme in action.  This is what the enzyme looks like as it goes  about its business.  Now what's hugely inefficient is the K off.  Do you remember earlier I told you the perfect enzyme  should have zero K off?  In this case, we see the enzyme in real time  in action being inefficient.  Here it is being inefficient as it goes one, two, one, two,  one, two.  This is it with the K off.  That catalytic inefficiency is what dooms the enzyme  and makes it waste opportunities.  The enzyme is trying to make up its mind.  Go to product, back to substrate.  Product, substrate, product, substrate.  And that lack of decision is what makes this enzyme inefficient  as a catalyst.  There are other kinases such as kinases involved  in metabolism that are far, far more efficient  than this enzyme over here.  If you're looking for something, could I ask you just  to wait until after the class is over?  Oh no, you're here for the class.  You're just kind of late.  Okay, no problem.  Welcome. All right.  So anyway, this is the enzyme in action.  And what this shows us is  that the enzyme fluctuates its speed enormously.  Okay? And this is kind of mind boggling.  And I'm just going to tell it to you.  It turns out that the enzyme's speed fluctuates  from second to second by a factor of 100.  Okay? So this is like going out to the 73 out here,  the freeway out here.  And then, you know, pulling up alongside a Honda Civic.  And then suddenly the Honda Civic goes from 55 miles an hour  to 5,500 miles an hour.  And then back down to 50 miles an hour all in the course  of a second.  So these enzymes are wildly changing their speeds.  They're changing speeds much faster than any runner.  They're changing speeds up to speeds that are wildly,  that are almost inconceivable to us humans.  And really, that's the stuff of life.  It's essential that this enzyme is able to alter its speed  in order for it to be regulated.  Remember earlier, I talked  about the regulation of protein kinase A. That regulation is  going to control its speed.  And in doing so, that turns the enzyme  from being a wildly efficient catalyst to being a catalyst  that's not even worthwhile, that doesn't operate  on a time scale that's useful for the cell.  And this is really the essence  of how catalytic biology takes place.  Okay? Any questions about this?  Yeah?  Do you see any pattern along that, the on, off,  or just randomly process?  Okay. This is a brilliant question.  We spent a lot of time looking for patterns in our data  and looking for correlation between one step or another.  And we, it sure is pretty random.  There is a small amount of a memory effect in the sense  that the enzyme, if it hits this intermediate state,  it's more likely to go down to three than it is to go back  to one.  Okay? And so that's actually a thermodynamic effect.  That the enzyme has evolved to do K-cat in preference to K-off.  All right.  Let's talk about another hydra,  let's talk about a different enzyme.  The second enzyme I want to talk, I want to talk  to you about today is lysozyme.  This is an example of a hydrolase.  And remember, I'm moving down, or actually I'm still  at the top up here in our most common enzymes.  This enzyme was discovered about 100 plus years ago  and it's been intensively studied.  This is the X-ray crystal structure of lysozyme.  And in fact, it was the very first enzyme,  X-ray crystal structure ever solved was this enzyme.  The active site up here has an 8-angstrom hinge motion.  And so this enzyme has kind of a Pac-Man-like motion  as it hydrolyzes the glycosidic bonds  of the polysaccharides found on the cell surface  of bacteria cells.  Okay? So here's a bacteria cell wall.  And the enzyme is going to cut apart the glycosidic bond  between each one of these glycan moieties found  on the cell wall.  Okay? So it's going to be chopping  apart the polysaccharide.  In doing this, this will basically burst apart the cell.  In the absence of this, the enzyme is basically going  to be chewing apart the bacteria.  And this has the effect of killing the bacteria, right?  You're breaking their cell walls.  They explode, et cetera.  This enzyme is found in high concentration in chicken eggs.  That's the hen egg whites over here.  And it's present to prevent colonization by bacteria.  And you might recall,  Avidin was isolated also from chicken egg whites.  So biochemists have been studying what makes eggs  so special as sterile vessels for a very long time.  Okay. Let's take a quick look at the arrow-pushing mechanism  for how this enzyme operates.  In this mechanism, the enzyme goes through a covalent  intermediate.  Let's start over here.  So I'm zooming in now to the polysaccharide region  of the cell wall of the bacteria.  Enzyme is going to cleave this bond that's indicated  with an arrow.  And this is an example of a hydrolase meaning it's going  to introduce water across this bond.  So the first thing that the enzyme does is torque this  anacetyl-maramic acid moiety.  Okay. So it's going to torque this carbohydrate  from being a nice chair conformation  to being a boat conformation.  This is a crucial aspect  for what makes enzymes such effective catalysts.  This enzyme is going to be catalyzing a reaction a thousand  times more efficiently than if the reaction just had  to happen by itself.  I actually think it's like a hundred thousand times more  efficiently.  In order to do this, the enzyme is going  to be physically bending the substrate.  And by physically bending the substrate,  this helps to accelerate the reaction.  So here it is pushed up into this boat conformation.  Notice that the boat conformation neatly sets up an SN2 attack  by this carboxylate of aspartic acid  to attack backside displacement style this glycoside.  Okay. This is crucial.  So the glycoside gets protonated by one glutamic acid  and then over here an aspartic carboxylate  from a nearby aspartic acid then attacks doing a backside  SN2 displacement.  And this protonated glycosidic bond then is a very effective  leaving group because the second arrow highlighted  in red over here kicks electrons  to a positively charged oxygen which is all too eager  to accept those electrons.  Okay. So this gives us a covalent intermediate.  This is another common way that enzymes accelerate reactions.  In this case we're seeing it form a covalent intermediate  and then this covalent intermediate gets hydrolyzed.  So half of the polysaccharide floats away.  The next one comes, a water comes in, gets deprotonated  by the glutamic acid, the glutamate up here.  And then this hydrolyzes the ester bond between this polysaccharide  and the aspartic acid.  Okay. So some notable features here.  We're showing, I'm showing you an example  of acid-based catalysis.  The enzyme is simultaneously acting as both an acid over here  and as a base over here.  In fact, it's even wilder than that.  Check this out.  It's the same functionality, this glutamic acid  that acts as both the acid and the base.  And to me that's just an elegant simplicity  that makes enzymes so beguiling, right?  If we were in the chemical laboratory trying  to make molecules, you know, using glassware and such,  we'd either dump in a bunch of acid or dump in a bunch of base,  but you wouldn't add simultaneously both acid and base  because they would neutralize each other.  And enzymes have evolved to be able  to simultaneously catalyze things using both acid  and base catalysis.  Furthermore, this enzyme has evolved to form a covalent  intermediate, sometimes this is referred to  as a ping-pong mechanism that eventually gives us back the  hydrolyzed glycosidic bond.  Okay, really, really beautiful.  This is what you learn in biochemistry classes.  And the problem with it is  that it neglects enzyme dynamics, which are really critical.  As the enzyme moves, it can then help to torque this confirmation  of the ring into this boat confirmation.  In the absence of this movement,  it doesn't make sense really why it is  that the enzyme is actually going to be torquing this substrate.  Right? Because the substrate binds  in the chair confirmation up here.  Why should it get pushed into this other confirmation  unless the enzyme is doing the pushing?  And that, in fact, is what we see.  Okay, so same idea.  We're going to attach an enzyme to the carbon nanotube  and then listen in as the enzyme works.  When we do that, here's a paper from the lab  from about a year ago.  What we see is that, again, the enzyme by itself is relatively quiet.  And then when we add the substrate, the polysaccharide,  the peptidoglycan that I showed on an earlier slide,  the crosslink net, there's an immediate jump upward  and then there's all this noise in here.  This is the enzyme chewing on the substrate  and we get to listen in.  It's just like SoundCloud, basically.  Okay, some controls.  I haven't been showing you controls,  but these are everything in biology.  This is substrate by itself in red, overlaid.  And then this is enzyme by itself.  Again, it's relatively flat and purple.  And then when we have enzyme plus substrate, we see this motion  where what we're seeing here is enzyme open, closed, open, closed,  open, closed, open, closed, open, closed, open, closed, open, closed.  This is it, you know, on a third of a second.  But we get to watch the same enzyme cranking over for a long period of time.  And again, what we find is that the enzyme is highly variable.  It accelerates, it slows down, it speeds up, it slows down.  It accesses different confirmations.  In fact, it accesses at least two dramatically different speeds.  And you can actually see that in the 40 seconds of data over here.  Do you see how there's this dense region and then a less dense region  and then a dense region?  The dense region corresponds to rapid switching.  The enzyme has an overdrive gear that it goes into.  So it flips gears to second gear and it just starts cranking along  at a much faster speed.  And you can see that over here where the enzyme is going, open,  closed, open, closed, open, closed, open, closed, open, closed, open,  like 300 times per second.  Whereas over here it's doing open, closed, open, closed, open,  closed like 50 times per second.  So this is dramatic.  That's six times faster.  And what's crazy is that the enzyme does this all day long.  It switches between first gear and second gear, first gear,  second gear, back and forth.  And a big mystery in the field is what's up with second gear?  Okay? So to address that question, I'm going to skip some stuff.  To address that question, together with collaborators,  we chemically synthesize a version of the polysaccharide  that didn't have cross links.  This is like one strand of the net that I showed you earlier.  Same polysaccharide.  Now we're using chemical synthesis to access a new substrate.  And what we find is that the enzyme has a different type  of activity.  I do have to show you some more controls.  Okay? I can't get away from this.  They're crucial.  These are mutant enzyme active sites.  Do you remember earlier I showed you the carboxylates  that are required for enzyme to operate?  So we mutated those carboxylate residues  and the enzyme no longer works and therefore it never closes.  The other one of these enzyme mutations traps the covalently  bound form of the substrate that I showed earlier  in the ping pong mechanism.  And the enzyme never can hydrolyze back off the substrate.  And again, it never gets back to closed.  Okay. So I get to finally tell you what the difference is  between first gear and second gear.  This is a day in the life of an enzyme.  Here's how it spends its time.  Okay? So this is an enzyme crank it along happily being fed  either the linear substrate or the crosslink substrate.  In the case of getting the crosslink substrate,  it gets to hydrolyze things about 50% of the time.  But if you feed it the linear substrate, it goes wild.  It gets to hydrolyze glycosinic bonds 88% of the time.  And so, and then this over here is second gear in blue.  That's nonproductive rapid chatter.  And over here in the crosslink, the blue is much more apparent.  So in other words, the crosslink substrate, the substrate found  on the surface of the bacteria cell, corresponds to second gear.  So what we think is happening is the enzyme is mowing across the  surface of the cell chewing contentedly.  Bond after bond after bond after bond, happily hydrolyzing it all.  And then it hits one of these peptide crosslinks and gets stuck.  And when it gets stuck, its response is to start chattering away.  It just, it flips gears and it just starts going six times faster.  And what we think is happening is that it then transits along the peptide  down to the parallel polysaccharide.  So in the same way that DNA is a five prime directionality  of three prime directionality, polysaccharides have a directionality as well.  And it turns out the surface of the bacteria cell is a highway  of parallel polysaccharides.  So the enzyme comes along, hits across link, goes down, zooms along,  hits crosslink, goes down, zooms along, down, cross, down, across.  So what the enzyme is doing is zigzagging across the surface of the cell  as it chews apart the surface of the bacteria.  And in retrospect, this totally makes sense because again,  the enzyme evolved to poke holes in bacteria.  And by doing a two-dimensional rip in the surface of the bacteria,  this makes the enzyme much more effective  at killing its bacterial targets.  Questions?  Yeah.  So one of the active UCs, the actual pylosys,  the N1 is it transporting itself?  Yeah. Well, it's a nonproductive chatter that we think is  that moving along one of these peptide crosslinks.  So, yeah, Anthony.  So as soon as it encounters something, it can't press through,  it'll switch back over.  Until it finds a new glycosidic bond and then it goes to town again.  Okay. Well, let's move on.  I want to talk to you about other enzymes.  We have lots to talk about.  I want to talk to you very briefly about proteases,  which cleave amide bonds.  We've seen these.  We've seen examples of these.  In blood, there's a whole cascade of proteases that are used  to respond to damaged blood vessels with a series of factor 7a,  factor 10a, et cetera.  Proteases where one cuts one and the next one cuts another,  next one cuts another, et cetera.  All the way down to the point where you get production  of fibrin, which then could crosslink to form to replace  to fix the damage up here.  Okay. So this is an important mechanism for blood clotting.  And naturally, if you're missing any one of these proteases,  or one of these proteases happens to be mutated,  you're in big trouble.  Your blood will not clot.  This happens in inbred families, such as the royal families  of Europe in the turn of the century.  This is the czar, Nicholas II.  His wife Alexandra passed on the gene for hemophilia  to their son Alexi down here.  And again, this is a mutation to either factor 9 or factor 8,  which are both excellent genes.  These are genes that are found on the X chromosome.  So they're passed along by the mother.  Okay. Apatosis is also regulated by a series of proteases.  Apatosis, the cell suicide mechanism that we talked  about earlier in this quarter.  And each protease activates the next one.  So you have a protease up here called the caspase  that cleaves the next caspase in line,  which then cleaves this, et cetera.  So this guy cleaves this guy, which cleaves this guy, et cetera.  Let's take a closer look at an example of a protease.  The protease I want to show you is one of my favorites.  It's isolated from one of my favorite fruits, papaya.  And it's perhaps appropriately called papine,  because it's isolated from papaya.  It's an example of a cysteine-based protease.  And it's furthermore another example  of a nucleophile-based enzyme mechanism.  And I chose this one because cysteine, of course,  is the preeminent nucleophile.  As illustrated earlier today when we talked  about engineering in a single cysteine on the surface  of the enzyme as a way of attaching it  to a specific spot on the carbon nanotube.  Okay, so here's the mechanism for how this enzyme works.  In practice, it's actually a fairly complicated mechanism,  sorry, it's a complex mechanism, in the sense  that it's a concerted mechanism.  Specifically, here's the nucleophilic file functionality  of the cysteine in the active site.  I don't think I pointed it out here, but here's the cysteine.  That's the business end of the molecule.  It is active site.  And in a concerted mechanism,  the cysteine is simultaneously deprotonated  to attack nucleophilically the amin bond.  And then this amin bond carbonyl gets protonated  by an acid residue that hovers above the carbonyl  of the amin bond.  This happens in one fell swoop from nucleophile  to protonation all at once.  And that is really that kind of concerted dance  of catalytic efficiency is yet another example  of what makes enzymes so special.  The fact that everything is kind of held together at once lowers  the transition state energy.  Right, so now you don't necessarily have  to stabilize a protonated carbonyl in an active site.  Instead, you wait until electrons appear up here  on this oxygen before it gets protonated.  So that lowers the transition state energy  for this transition state.  Okay, there's other ways of depicting this as well.  I prefer this one concerted mechanism.  So all of these, okay, actually I'm going to skip the serine  base proteases.  They're similar to the cysteine base protease I showed earlier.  I'm going to skip the zinc proteases as well.  There's quite a few others.  Like the kinases, the proteases, because they're involved  in crucial processes in human physiology such as blood clotting  which you would not want to happen, you know, here and there,  because these are involved in such crucial processes,  the enzymes themselves are tightly regulated,  oftentimes regulated by some loop in a pro enzyme  that has to be cleaved.  Where the terminology of pro means a reaction takes place  that then converts it into the active functionality  of the molecule.  So for example, pro drugs are precursor drugs  that are then converted into the active drug  by some enzymatic process.  And over here, we see a pro enzyme that has a loop blocking access  to the active site of the enzyme that gets cleaved  and then that allows the pro enzyme piece to dissociate  and turn on the enzyme.  So enzymes can be very readily inhibited.  You can do things like have transition state analogs.  We've talked about transition state analogs before.  Here is the transition state for hydrolysis of an amine bond  and here is a very effective phosphor amide transition state.  Notice that this also has the tetrahedral geometry  of this transition state up here.  And if you do that, you can actually very effectively  inhibit this enzyme.  Other types of inhibitors, phosphonates down here,  phosphor amides over here, these KIs are the dissociation  constant for binding where it's KD except it's  for inhibiting, binding and inhibiting the enzyme.  And again, smaller numbers equals more potent enzymes.  And notice what a champion phosphor amide is  with a picomolar, near picomolar inhibitor.  Okay. Now, there's a million other things I could talk  to you about.  I'm going to pick them up next Thursday.  When we come back, we'll be finishing off Chapter 6  and going on to Chapter 7.  Midterm we'll cover through today's lecture.
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