Lecture 13 Summary

We note that from an experimental standpoint, k_cat and k_cat/K_M are the fundamental parameters. The turnover number, k_cat, is measured under conditions in which the enzyme is saturated with substrate (very high [S] or "V_max" conditions), while k_cat/K_M is best measured at low substrate concentrations (where [S] << K_M, which we can call "V/K" conditions).

We discuss the range of values observed for both K_M and k_cat, also noting the distinction between a high turnover number (high k_cat) and the large rate enhancement (high k_cat/ k_uncat - see Table 8.1 on p.206.) of an enzyme-catalyzed reaction over the uncatalyzed rate for the same reaction. Recall the key concept from this chapter: Enzymes achieve such tremendous acceleration of reaction rates by lowering the free energy of activation for the reaction. Exactly how enzymes stabilize the transition state is a fascinating area of study. The quantity k_cat/K_M was then discussed as a measure of specificity (in comparing the efficiency with which an enzymes acts on a series of closely related substrates) and as a criterion for so-called "catalytic perfection" in which k_cat/K_M approaches a theoretical upper limit (the diffusion-controlled limit).

Moving beyond kinetic parameters, we spend some time considering mechanistic schemes involving reactions with multiple substrates, e.g. an enzyme catalyzing a reaction such as A + B <—> P + Q. The text discusses sequential (both the ordered sequential and random sequential mechanisms) and double-displacement ("ping-pong") schemes. We also see that in some cases enzymes display non-Michaelis-Menten kinetics and that a primary example of this is the case where the initial velocity vs. substrate concentration curve is sigmoidal rather than hyperbolic. This behavior occurs with multisubunit enzymes that have allosteric properties arising from cooperativity between subunits.

Finally we introduce the topic of enzyme inhibition by considering two idealized simple models for the phenomenon, (see Fig.8.15, p.225) distinguishing them by the terms competitive inhibition and noncompetitive inhibition. Enzyme inhibitors are not only important tools for enzymological research, but also more broadly significant in biochemistry, biology, and medicine. Many drugs, for example, are specific inhibitors of particular enzymes. Aspirin is an irreversible inhibitor of cyclooxygenase activity.

Enzyme inhibition

The specificity of enzymes is not strictly limited to substrates. Often, the activity of an enzyme is reduced by specific interactions with molecules termed inhibitors. Enzyme inhibition is one of the most important phenomena in biochemistry. For example, many drugs, antibiotics, and toxins exert their effects by ther ability to inhibit an enzyme. Inhibitors that are structurally similar to the substrate are often competitive inhibitors, since they compete for binding at the active site. Enzyme inhibiton can be reversible (as is usually the case when an inhibitor binds to the enzyme via noncovalent interactions) or irreversible (as occurs in numerous cases where inhibitors act via covalent modifications to the enzyme, perhaps targeting a critical residue for catalysis).

We can imagine several simple models for reversible inhibition. The simplest of these is the direct occlusion of the active site by the inhibitor. This would be seen in the case of a molecule with some structural similarity to substrate. Binding of substrate and inhibitor are mutually exclusive in this model for competitive inhibition.

By increasing [S] to ever greater levels, the substrate can overwhelm the inhibitor, outcompeting it for the free enzyme to the extent that the true V_max can still be approached.

Types of reversible enzyme inhibition - a summary

We have seen that the different models for reversible inhibition can be distinguished according to effects on kinetic parameters. The table below summarizes the types of inhibition and their effects on these parameters.

Learning objectives

• Distinguish between "V_max" and "V/K" conditions .
• Define inhibitor, and describe the molecular basis for the effects of inhibitors.
• Define and distinguish between the following types of inhibition:
  • Competitive inhibition
  • Noncompetitive inhibition
  • Uncompetitive inhibition
  • Mixed inhibition
  • Irreversible inhibition (distinguish from reversible inhibition)

Page updated 10-11-09

References

1. Cornish-Bowden, A. Fundamentals of Enzyme Kinetics (Revised ed. 1999, Portland Press)
2. Fersht A. Structure and Mechanism in Protein Science (1999, WH Freeman and Co.)
3. Jencks WP. Catalysis in Chemistry and Enzymology (1987, Dover)
4. Antibodies Molecule of the Month @ Protein Data Bank (this MOM feature includes an example of a catalytic antibody).