e reaction can occur more easily. This speeds up the rate of the reaction as it allows the product to be formed faster.
An enzyme has a high affinity for the transition state (even higher than for its substrate). Therefore when the substrate binds, it is quickly forced into the transition state. This is a state that exists between the substrate and the product. The enzyme is said to facilitate the formation of the transition state.
The transition state has a high energy, making it very unstable. It can only exist transiently. The transition state spontaneously turns into the more stable product with lower energy. The enzyme will have a low affinity for the product and so the product is released.
Rate Limiting Steps
The rate limiting step in any reaction is its slowest step. It sets the pace for the entire reaction. After all, a production line can only be as productive as its slowest worker. In enzymatic reactions, the conversion of the enzyme-substrate complex to the product is normally rate limiting. The rate of this step (and therefore the entire enzymatic reaction) is directly proportional to the concentration of ES.
The concentration of ES changes as the reaction progresses. Therefore, the rate of product formation also changes over time. When the reaction reaches equilibrium (steady state) the concentration of ES (and therefore the rate) remains relatively constant.
When an enzyme is added to a lot of substrate, the reaction that follows occurs in three stages with distinct kinetics:
|Phase||Concentration of ES||Rate of product formation|
|Pre-steady state||Burst of ES complexes form||Slow as must first wait for ES to form. Speeds up as ES forms.|
|ES remains constant. It is formed as quickly as it breaks down.||Constant rate of formation. Faster than pre-steady state.|
|Post-steady state||Substrate depletes so fewer ES complexes can form||Slow as there are fewer ES complexes. Slows down as substrate runs out.|
The pre-steady state phase is very short as equilibrium is reached within microseconds. If you measure the rate in the first few seconds of a reaction (V0) you are actually measuring the steady state. This is the rate used in Michaelis-Menten Kinetics.
Two terms that are important within Michaelis-Menten Kinetics are:
- Vmax – the maximum rate of reaction when all enzyme active sites are saturated with substrate
- Km – the substrate concentration that gives half maximal velocity. Km is a measure of the affinity an enzyme has for its substrate, as a lower Km means that less of the substrate is required to reach half of Vmax.
This equation concerns the steady state of an enzymatic reaction with one substrate, and is given by:
It describes how the initial rate of reaction, V0, is affected by the initial substrate concentration, [S]0. It only looks at the start of the reaction. This allows it to ignore the reverse reaction where substrate is formed from product. This is because at the start of the reaction there is no product present to become substrate.
The plot of rate against concentration has the shape of a rectangular hyperbola. However, a more useful representation of Michaelis–Menten kinetics is a graph called a Lineweaver–Burk plot. The equation used to generate this plot is given by:
This allows an easier interpretation of various quantities from the graph, such as the presence of an inhibitor. This is shown in figure 3, where an inhibitor decreases Km and therefore shifts the original line upwards.
Clinical Relevance – Plasma Enzyme Assays
Plasma enzyme assays can detect abnormal levels of enzymes in plasma. Levels may be abnormal due to leakage from damaged tissue or lack of functional enzyme production. The assay measures units of activity in a sample and so it will not measure non-functional enzyme.
If levels of an enzyme are raised it may indicate damage to the tissue that enzyme is normally found in.
If levels are low it may indicate that the enzyme is either non-functional, being produced slower or broken down quicker possibly due to a genetic abnormality.
|Enzyme||Location||Indication if raised|