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Enzyme inhibitor mechanism Decrease the enzyme ability to bind substrate.

Lowers the enzyme catalytic activity Do both the above. There are two main classes of enzyme inhibitors, reversible and irreversible, that are differentiated by the magnitude of their affinity for enzyme. Reversible enzyme inhibitors bind and dissociate with their enzyme in a equilibrium process. Irreversible inhibitors bind tightly to an enzyme to form an essentially permanent complex. RREVERSIBLE INHIBITORS Because they form such a tight complex with the enzyme that they permanently remove its catalytic activity, irreversible inhibitors are also termed inactivators. For example, kinetic studies on the male pattern baldness drug finasteride (Propecia) reveal it to be an essentially irreversible inhibitor of the 5a-reductase. This enzyme catalyzes one step in the conversion of testosterone to dihydrotestosterone, which is a hormone implicated in hair loss.

Some irreversible inhibitors even become covalently bound to amino acids in the active site of the enzyme as they are brought through the early chemical steps of catalysis. Aspirin is such an example. REVERSIBLE INHIBITORS Reversible inhibitors can be classified as competitive, mixed, or noncompetitive inhibitors. If the detailed mechanism of inhibition is known, then the classification can be made by identifying where on the enzyme the inhibitor binds, or the order with which it binds, relative to substrate. Alternatively, a determination of simple kinetic parameters can generally be used to classify the inhibitor. COMPETITIVE INHIBITION Competitive inhibitors compete with substrate for an enzymes active site, lowering the enzymes likelihood of binding substrate and slowing the observed reaction velocity. Competitive kinetics Kinetic studies can be used to determine the type and potency of inhibition for an unknown inhibitor. Typical steady-state kinetic experiments can be performed where reaction velocity is measured in the presence of varying concentrations of substrate. If inhibitor is then added, and the data shows an increase in KM, yet the Vmax is unaffected, this is the signature of a competitive inhibitor.

MIXED INHIBITION A mixed inhibitor binds to a site on the enzyme and interferes with both apparent substrate affinity and catalytic turnover, thus affecting both the observed KM and kcat for the enzyme-catalyzed reaction. Mixed inhibitors do not bind directly in the active site, and therefore do not block substrate binding, but instead bind at sites that can be proximal or distal from the active site. Mixed inhibitors can therefore bind to free enzyme prior to substrate, distorting the active site to a nonoptimal conformation for catalysis. The inhibitor-distorted active site has trouble converting the substrate to product before it dissociates, resulting in a lowered apparent substrate binding affinity. Mixed inhibitors distort the active site, interfering with the chemistry performed by the enzyme. Therefore the enzymatic turnover rate is slowed. Mixed inhibitors can also bind after substrate to the enzyme-substrate complex. Similar to before, the inhibitor binding to the ES complex distorts the active site and its bound substrate to a nonoptimal conformation, so that less ES complex productively form product before the substrate dissociates. Binding of the mixed inhibitor to the ES complex also interferes with the subsequent chemistry step. Mixed kinetics Steady-state experiments performed in the presence of a mixed inhibitor demontrate an increase in KM, and a decrease in Vmax. Remember, Vmax is simply kcat multiplied by the total enzyme concentration. Therefore, a decrease in kcat is a decrease in Vmax. NONCOMPETITIVE INHIBITION Noncompetitive inhibition is a special case of mixed inhibition where the affinity of inhibitor for E and ES is the same. Noncompetitive kinetics Steady-state experiments performed in the presence of a noncompetitive inhibitor demonstrate a decrease in Vmax, yet KM is unaffected.

ffects of Inhibitors on Enzyme Activity

Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition competitive, non-competitive and substrate inhibition.

Most theories concerning inhibition mechanisms are based on the existence of the enzyme-substrate complex ES. As mentioned earlier, the existence of temporary ES structures has been verified in the laboratory.

Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. A theory called the "lock-key theory" of enzyme catalysts can be used to explain why inhibition occurs.

The lock and key theory utilizes the concept of an "active site." The concept holds that one particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in such a way that its conversion to the reaction products is more favorable. If we consider the enzyme as the lock and the substrate the key (Figure 9) - the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds. However, when an inhibitor which resembles the substrate is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds normally.

Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate. Figure 10.

Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 11 shows the reaction velocity decreasing after the maximum velocity has been reached.

Additional amounts of substrate added to the reaction mixture after this point actually decrease the reaction rate. This is thought to be due to the fact that there are so many substrate molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 12) and prevent any other substrate molecules from occupying them.

This causes the reaction rate to drop since all of the enzyme present is not being used.

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