Biochemistry

Lecture 15: Enzyme Inhibition

Assigned reading in Campbell: Chapter 5.7- 5.10

Take the Review Quiz on Lecture 15 concepts.
Data Analysis for Competitive Inhibition The class handout shows enzyme inhibition curves.

Structure of a Trypsin-Inhibitor Complex: "Must-viewing" of an enzyme-inhibitor complex on a Chime page.
An Oxygen Binding Simulation that shows the differences between the "concerted" and "sequential" mechanisms of allosterism applied to hemoglobin.

Please note: The shaded sections of these revised lecture notes will not be covered this term.
10.13.00

5.7 Inhibition of Enzymatic Reactions

Studies on Inhibitors are useful for:

1. Mechanistic studies to learn about how enzymes interact with their substrates.
2. Role of inhibitors in enzyme regulation.
3. Drugs if they inhibit abberrant biochemical reactions:
• penicillin, ampicillin, et al.: interfere with the synthesis of bacterial cell walls.
• methotrexate: anti-cancer drug that affects DNA metabolism in actively growing cells
• viagra: interferes with nitric oxide breakdown (NO is a vasodilator)
4. Understanding the role of biological toxins.
• Arsenate: mimics phosphate esters in enzyme reactions, but are easily hydrolysed.
• Amino acid analogs: useful herbicides (i.e. roundup)
• Insecticides: chemicals targeted for the insect nervous system.

A. Competitive Inhibition

1. Inhibitor binds to the same site on the enzyme as the substrate, the active site.
2. Inhibitor only binds to the free enzyme.
3. Inhibitor is usually structurally very similar to the substrate. Examples:
• Succinate/Malonate
• ATP/AMP

The reaction scheme that corresponds to competitive inhibition is:

There are two anticipated consequences of this additional competitive equilibrium:

1. V_max is unchanged: At high levels of substrate all of the inhibitor is displaced by substrate.
2. K_M is increased: Higher substrate concentrations are required to reach the maximal velocity.

Steady-state analysis of the effect of the inhibitor shows that K_M is increased by a factor of (1 + [I]/K_I). The resulting form of the Michaelis-Menten equation is:

Measurements of complete saturation curves (i.e. v_o versus [S]) at different inhibitor concentrations can be used to obtain the dissociation constant for inhibitor binding. The Lineweaver-Burk plots will show an unchanged V_max and a slope that increases with inhibitor concentration. (Figure 5.10). Campbell's equation (5.18) for the double reciprocal plot shows that the slope of the lines will be:

Thus, K_I can be determined by plotting the slope values vs. [I]. The resulting secondary plot or "replot" will have a Y-axis intercept of K_M/V_max and a slope of K_M/(V_maxK_I). The value of K_I is the slope/intercept of this replot.
The lecture handout, Data Analysis for Competitive Inhibition shows an example of the use of replots to determine the K_I values.

B. Noncompetitive Inhibition:

In this case the inhibitor binds to both E and ES. Both the slope (K_M/V_max), and the Y-intercept (1/V_max) of the Lineweaver-Burk plot increase (see figure 5.11). The K_I ('s) are determined as above by replotting the slope and intercept values vs. [I].

1. V_max is decreased: At high levels of substrate the inhibitor is still bound.
2. K_M is increased: Higher [S] is required to reach the lower maximal velocity. (For "simple noncompetitive inhibition", K_M is not changed, e.g. Fig. 5.11 in Campbell.)

Irreversible Inhibition

In contrast to the above types of reversible inhibition, where the effects on the enzyme reaction depend on the concentration of an inhibitor (and its K_I), there are many examples of compounds that react chemically with residues in the enzyme active site. In these cases, enzyme activity is destroyed. For example, the "nerve gas" Sarin reacts specifically with an active site Ser residue on the enzyme, acetylcholinesterase. If acetlycholine cannot be hydrolyzed by this enzyme, nerve signals cannot be passed across the synapses of the nervous system. On exposure to this compound, death can result in minutes due to respiratory failure.

5.8 "The Michaelis-Menten equation does not describe the behavior of allosteric enzymes."

The above title statement, quoted from Campbell, is misleading. What is true is that the simplest form of the Michaelis-Menten equation does not account for the higher than first order substrate concentration dependence found in many allosteric enzymes. But this is the same as saying that the Scatchard equation does not describe O₂ binding by hemoglobin. In each case, the basic concept of saturation (of an enzyme active site or a ligand binding site) is identical. In fact, the cooperativity observed in allosteric enzymes is analyzed using the Hill equation and Hill plots to obtain values for K_M and n_H.

Allosteric enzymes are usually found at metabolic control points. Frequently, these are the first steps in pathways. Regulation of the entire sequence of enzymatic steps is most efficiently accomplished by turning on or turning off the pathway at the first step. When the control is exerted by the end product of the pathway, the inhibition (or activation) is termed "feedback regulation". An example to be considered later is the feedback inhibition by ATP of the glycolytic pathway. Allosteric regulation always involves the binding of a metabolite to a site separate from the enzyme active site. In this regard, the regulation is analogous to the allosteric effects of BPG on O₂ binding by hemoglobin.

5.9 Models for the behavior of allosteric enzymes

The concerted and sequential models for describing allosteric behavior are discussed in detail by Campbell. Although the terminology has permeated the biochemical literature and is widely (over-)used, bear in mind that neither model accurately describes any real enzyme-substrate or protein-ligand interaction! Stated another way, it has not yet been possible to obtain data of sufficient accuracy to distinguish between the two models. The most intensely studied system is hemoglobin, where it now appears that a mixture of the two models may be the best (current) description.

5.10 Zymogens

Campbell's introduction to the topic of proteolytic processing provides the basic concepts of a frequently-observed regulatory mechanism in metabolism and in the differentiation and development processes of organisms.

5.11 - 5.13

We will return to these topics at a later date in the semester (after substitution and elimination reactions have been treated in Organic Chemistry).

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