There are numerous enzymes which have been identified as capable of catalyzing various chemical reactions. Similarly, it has been discovered that antibodies can be elicited to catalyze a variety of chemical reactions (U.S. application Ser. No. 674,253). It is well known that antibodies and enzymes share a fundamental similarity in that both are specialized proteins that bind to other molecules. However, there are important differences between antibodies and enzymes.
Antibodies typically bind to a molecule or antigen so that the antigen is marked as foreign to the organism that produced the antibody. The binding of the antibody to the antigen enables the antigen to be removed from the organism. Enzymes are biological catalysts which bind a molecule in such a way that the activation energy of a reaction involving a molecule or substrate is lowered, thereby increasing the rate of the reaction.
Linus Pauling discovered there are two types of interactions between proteins and the molecules that bind them. Antibodies bind molecules in their ground state while enzymes bind molecules in higher energy states.
Pauling attempted to explain the mechanism of enzyme catalysis based upon such binding. During the course of the chemical reaction, the reactants undergo one or more transitions through intermediate structures or transition states which are energically less favorable than either the reactant or the product. The hydrolysis reaction of a peptide linkage or an ester bond in an aqueous medium passes through a tetrahedral carbon transition state, as depicted in FIGS. 1 (peptide) and 2 (ester). In the transition state, a tetrahedral carbon atom is bonded to: a carbon atom of the acid portion of the peptide linkage or ester bond; two oxygen atoms, one corresponding to the carbonyl group and the other corresponding to a hydroxyl ion or water molecule of the medium; and either the oxygen atom of the alcohol portion of an ester or the nitrogen atom of the amine portion of the peptide linkage. The transition state can be neither isolated nor detected since it exists for only about 10−13 sec.
In molecular terms, these transition states reflect changes in bond lengths and bond angles as well as bond formations and breakages. The energy required to achieve a transition state is denoted as the activation energy which may also be considered as the difference in energy between the energy of the transition state and the energy of the reactants. According to Pauling's explanation, an enzyme preferentially binds the transition state of a reaction, thereby stabilizing it relative to the substrate and products and reducing the activation energy of the reaction, thus increasing the reaction rate. For example, aspartic proteinases are enzymes which are known to catalyze the hyrolysis of peptide linkages within a protein molecule.
By extending this explanation, Pauling also predicted that stable analogs of a transition state would bind tightly to an enzyme. In a discussion of substrate distortion as one of several possible sources of rate enhancement by enzymes, it has been suggested that the term “transition state analog” might be used to describe an inhibitor of this kind (1a).
The expression of binding energy to compensate for geometrical strain in the substrate is a popular but unlikely mechanism of catalysis. The intermolecular force field cannot overcome the intramolecular force field of the substrate (1).
Compensation of unfavorable solvation changes by the binding energy is also a possible mechanism of catalysis because of the large solvation energies of the polar groups and ions in water. Lone pairs which may act as general bases or nucleophiles will usually be solvated by hydrogen bonding from water or enzyme. Similarly, other reactive groups will usually be “neutralized” by hydrogen bonding or other mechanisms in the initial state of the enzyme. Usually these groups must be “desolvated” before reaction can occur. This process is energetically expensive and yet an essential part of the normal activation energy which may be compensated by favorable interactions between the non-reacting part of the substrate and the enzyme.
The expression of binding energy to compensate for unfavorable electrostatic interactions is another possible mechanism of catalysis. The juxtaposition of like charges could occur in the initial state but be removed in the transition state (2).
Pauling's prediction has become the basis for the now well-established approach to enzyme inhibitor design. The strategy for designing enzyme inhibitors has suggested a strategy for preparing catalytic antibodies whereby antigens are designed based upon mechanistic principles so that antibodies raised in response to such antigens will catalyze a chemical reaction by carrying out the reaction mechanism implicit in the design of the antigen. This strategy has been attempted a number of times.
For example, a transition-state analog mimicking an intramolecular 6-member ring cyclization transition state was used to elicit a monoclonal antibody which acted as a stereospecific, enzyme-like catalyst (3). Specifically, the monoclonal antibody so elicited accelerated, by about a factor of 170, the formation of a single enantiomer of a δ-lactone from the corresponding racemic δ-hydroxyester.
Similarily, monoaryl phosphonate esters, designated as analogs of the transition state in the hydrolysis of carboxylic esters, were synthesized and used as haptens to elicit specific monoclonal antibodies capable of catalyzing the hydrolysis of carboxylic esters (4). Certain of the antibodies elicited were reportedly found to be catalytic and selective for the hydrolysis of particular aryl esters.
Phosphonamidates or phosphonate analog-ligands having conformations that substantially correspond to the conformation of a hydrolytic transition state of an amide or ester ligand and which have been used to produce antibodies are described in U.S. Pat. No. 4,659,567 to Tramontano et al. (Tramontano). Antibodies so produced include a paratope that binds to and stabilizes the tetrahedral carbon atom of the amide or ester hydrolysis transition state of the ligand to hydrolyze the ligand at a predetermined site.
Analog-ligands which can be used to produce antibody catalysts for the hydrolysis of esters and amides are also described in European Patent Application 0,251,093 of Kollmorgen Corp. (Kollmorgen).
However, none of these analog ligands have been designed in accordance with a rational design approach which maximizes stabilization of the transition state and optimizes atomic relationships within the proteolytic transition state analog, thereby enabling the elicitation of antibodies capable of producing the two dramatic effects of enzyme catalysis; these are molecular recognition and rate acceleration.
In enzyme catalysis, groups on both the substrate and the enzyme which are not involved in the chemical mechanism of bond making and breaking, make an important contribution to catalysis. This is illustrated by examining a system where the mechanism is the same for both the enzyme and the non-enzyme catalyzed reaction. For example, the mechanism of action of succinyl-CoA acetoacetate transferase involves nucleophilic attack of the enzyme's glutamate carboxylate on the thioester succinyl CoA, [1] to give an anhydride intermediate (5). The second order rate constant for this reaction is 3×1013 fold greater than the analogous reaction of acetate with the same ester [2]: 
Although the nucleophilicities of the carboxylates may be slightly different because of solvation effects and the enzyme may provide some other forms of catalysis these contributions will not be large. The non-reacting part of the enzyme therefore lowers the activation energy by up to 78 KJmol−1. Similarly, changing part of the substrate structure away from the atoms involved in bond making and breaking may also significantly effect catalytic efficiency. The enzyme forms anhydride intermediates from “non-specific” substrates [3]. Even though the chemical reactivities of the two substrates, [1] and [3], are similar, e.g. towards alkaline hydrolysis, the enzyme reaction proceeds up to 3×1012 fold faster with the so called specific substrate [1](5). The non-reacting part of the substrate—the CoA residue—lowers the activation energy by 72 KJmol−1 compared with [3].
Thus, the haptens disclosed in Tramontano do not provide the correct architecture to elicit antibodies that are capable of catalyzing the cleavage of a predetermined peptide sequence in a native protein. These haptens do not provide the correct side-chain groups for production of antibodies that can react with predetermined sites on a protein and cause selective proteolysis in a sequence specific manner. Furthermore, these haptens do not incorporate amino acid side-chain sub-sites on either side of the transition state analog. Without these sub-sites, the haptens cannot provide for the elicitation of catalytic antibodies capable of recognizing a specific amino acid sequence and selectively proteolyzing a peptide linkage within that sequence.
Moreover, the haptens disclosed in Tramontano do not incorporate leaving groups which facilitate and thereby promote proteolytic catalysis. FIG. 3 (6) depicts a representation of the expected free energy diagram for serine proteinase catalysis. In this mechanism, the enzyme provides a nucleophile (Ser-195). A mechanism involving only a single transition-state requires concerted general base/general acid catalysis by the imidazole (His-57) of the catalytic triad in which the hydrogen from Ser-195 is shared between the serine (Ser 195), imidazole (His-57) and a leaving group, an unlikely situation. Given that a discrete TI exists, there must be two transition states along the acylation pathway (as shown in FIGS. 3 and 4).
The stabilization of the carbonyl oxyanion by hydrogen bonds from the protein, the so called “oxyanion hole” in the serine proteinases, is clearly a critical component of the enzyme's catalytic apparatus. The key residue Asn-155 involved in this hydrogen bonding network has been modified by site-directed mutagenesis and results in a decrease of 2–3 orders of magnitude in Kcat, with little change in Km. Decreases in transition state stabilization of 2.2 to 4.7 Kcal mol−1 were observed.
The imidazole of His-57 plays two main roles in the acylation step; first, as a base to facilitate attack by the Ser hydroxyl in formation of the tetrahedral species; and second, as an acid in protonating the leaving group in formation of the acyl-enzyme. As shown in FIG. 5, the TI/imidazole system can exist in four ionization states, including the zwitterionic form [2] in which both the imidazole and the carbonyl oxygen are charged. Only the TI species [2] and [3] can directly give transition states leading to the acyl-enzyme. These transition states differ from one another in that the carbonyl oxygen atom is either protonated [3] or in the oxyanion form [2]. Factors involved in determining the actual reaction pathway include the relative pk's of the amine leaving group nitrogen, the serine oxygen and the carbonyl oxygen.
For example, an unprotonated amine nitrogen will be a much poorer leaving group than the serine in a tetrahedral transition state (compare pk's of approximately 30 for the former against 15 for the latter). Thus the zwitterionic forms of TI and TS should be less stable, but more efficient at expelling the leaving groups than the corresponding neutral forms. Consequently, the zwitterions are expected to be the catalytically relevant forms (7). Electrostatic interactions within the enzyme's active site will contribute significantly to stabilizing the zwitterionic species.
It has been estimated that the pk of the leaving group nitrogen for amide substrates is between 8 and 11 in species such as Tl2 (8). Thus, if the pk of the imidazole remains around 7 in the transition state, then the leaving group has a higher proton affinity than the imidazolium (9). Accordingly, breakdown to the acyl-enzyme would be expected prior to complete formation of the TI. Several lines of evidence, utilizing transition state analog inhibitors, suggest that the true pk of the imidazole in the tetrahedral species is much higher than 7. Although the discussion has centered on the acylation half of the reaction (which is usually the rate-limiting step for amide and peptide substrates) the principles regarding the transition state and tetrahedral intermediate are valid for the deacylation step as well.
Thus, for example, the aromatic compounds described in Tramontano do not lend themselves to provide antibodies that catalyze the hydrolysis of amide bonds in peptides or protein sequences since the pk's of these compounds are significantly more acidic (aniline pka=4.6; p-methylaniline pKa=5.0) and the transition state stabilization afforded by antibodies raised to these immunogens will not provide for hydrolysis of peptide amide bonds since the actual reaction pathways are different.
Moreover, catalysis occurs through stabilization of the transition state of a reaction. As described below, destabilization of the ground state enzyme/substrate complex relative to the transition state is also necessary for enzymatic catalysis; this prevents accumulation of the enzyme in the enzyme/substrate complex (compare antibody/antigen complex). As noted in Tramontano, it has been suggested that the induced fit mechanism can aid catalysis by providing ground state destabilization relative to the transition state. However, while the induced fit mechanism can increase the value of Km, which reflects the energy of enzyme/substrate complexes, no catalytic advantage is provided for the following reasons.
The requirement for destabilization of the enzyme/substrate ground state is illustrated by the reaction catalyzed by two hypothetical enzymes E in FIG. 6 (10,11). Referring to FIG. 6, curve A shows the energetics for the uncatalyzed reaction of substrate S in solution. Curve B shows the reaction when the enzyme stabilizes the ground state and the transition state (ES and ES↑) to the same extent. The enzymatic ground state, ES, is more stable than free S by the amount GB, and the transition state S by the same amount. For the enzymatic reaction depicted by curve B, the rate limiting step is ESEP, and the energetic barrier for this step is ΔGA. This is the same barrier as for the reaction of free S. Thus the enzyme depicted in curve B does not catalyze the reaction, even though the transition state is stablized by this enzyme.
Still referring to FIG. 6, curve C shows what the enzyme (or antibody) must do; i.e., the transition state complex (ES) must be stabilized as in curve B but the ground state complex (ES) must also be destabilized relative to that in curve B by the amount ΔGB. The energetic barrier for the reaction in curve C, ΔGC, is much less than the barrier for free S (curve A/ΔGA) or the barrier for the enzyme of curve B (also ΔGA). The destabilization of the ground state relative to the transition state that is brought about by the enzyme of curve C is necessary for catalysis. This destabilization corresponds to an increase in Km while the value of Kcat/Km remains constant.
Enzymatic catalysis will be increased by increasing the energy of the ES complex as long as the transition state energy is not also increased; again, this corresponds to an increase in Km at constant Kcat/Km(12,13). The enhanced efficiency from increasing Km can be explained as an advantage in minimizing the amount of enzyme tied up as ES and thus maximizing the amount of enzyme available for catalysis of the reaction: E+SE+P. The enhancement in the observed rate by increasing Km at constant Kcat/Km is large when the substrate concentration is near or above Km because enzyme, which is complexed with substrate when Km is low, becomes available for catalysis. When the substrate concentration is well below Km a further increase in Km at constant Kcat/Km will yield no advantage because essentially all the enzyme is free and already catalyzing the reaction with the apparent second order rate constant Kcat/Km.
The fact that the second order rated constant for reacton of free enzyme and free substrate is Kcat/Km can be derived from the Michaelis-Menten equation:   ν  =                                          K            cat                    ⁡                      [            S            ]                          ⁡                  [          E          ]                    ⁢      tot                      [        S        ]            +              K        m            
At low substrate concentration ([S]<<Km), all of the enzyme is free and the Michaelis-Menten equation reduces to ν=(Kcat/Km) [S][E]tot. At high substrate concentration ([S]>>Km), all of the enzyme is in the form ES and the Michaelis-Menten equation reduces to ν=Kcat[E]tot.
Although an increase in Km at constant Kcat/Km is useful for catalysis, the induced fit mechanism increases Km but decreases Kcat/Km, in fact lessening catalysis (13). The decrease is Kcat/Km for the induced fit enzyme relative to the non-induced fit enzyme arises because some of the energy that is used to stabilize the transition state for the non-induced fit must be used to drive the conformational change of the induced fit enzyme. An equivalent explanation is that the decrease is Kcat/Km arises from a lower concentrtion of the active enzyme conformation in the induced fit mechanism (14).
The mechanism by which antibodies both destabilize the ground state complex and express intrinsic binding energy for the transition state of the particular reaction to be catalyzed has not been completely understood. An induced fit mechanism for antibody-antigen interaction has been proposed for a monoclonal anti-fluorescein-fluorescein hapten system (15). The binding of the antibody to the hapten was found to display a two-step mechanism; formation of an encounter complex followed by a tightening of binding to the hapten in an “induced fit” mechanism allowing for suitable contact interactions between the antibody combining site and the bound fluorescein ligand. If such a mechanism operates in binding of antibodies to their respective antigenic ligands then there exists an analogy between antibody/antigen interactions and enzyme/substrate interactions.
In order for antibodies to function as catalysts, design of the immunogen is crucial for allowing expression of binding energy for the transition state of the reaction. In order for antibodies to destabilize the antibody substrate complex, some form of conformational control of the immunogen is required which will allow for the production of strain and/or distortion of the substrate towards a transition-state trajectory in energetic terms.
Therefore, the immunogens described in Tramontano and Kollmorgen do not incorporate all these necessary features to provide for antibodies having catalytic function because they have not been rationally designed from knowledge of the mechanistic features of enzyme catalysis. Accordingly, they do not provide suitable templates for generating antibody combining sites endowed with catalytic properties.