Enzyme reactions reversible inhibition patterns

TABLE 11.6 Reversible inhibition patterns of enzyme reactions... 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

In practice, uncompetitive and mixed inhibition are observed only for enzymes with two or more substrates—say, Sj and S2—and are very important in the experimental analysis of such enzymes. If an inhibitor binds to the site normally occupied by it may act as a competitive inhibitor in experiments in which [SJ is varied. If an inhibitor binds to the site normally occupied by S2, it may act as a mixed or uncompetitive inhibitor of Si. The actual inhibition patterns observed depend on whether the and S2-binding events are ordered or random, and thus the order in which substrates bind and products leave the active site can be determined. Use of one of the reaction products as an inhibitor is often particularly informative. If only one of two reaction products is present, no reverse reaction can take place. However, a product generally binds to some part of the active site, thus serving as an inhibitor. Enzymologists can use elaborate kinetic studies involving different combinations and amounts of products and inhibitors to develop a detailed picture of the mechanism of a bisubstrate reaction. 

Reversible inhibition occurs rapidly in a system which is near its equilibrium point and its extent is dependent on the concentration of enzyme, inhibitor and substrate. It remains constant over the period when the initial reaction velocity studies are performed. In contrast, irreversible inhibition may increase with time. In simple single-substrate enzyme-catalysed reactions there are three main types of inhibition patterns involving reactions following the Michaelis-Menten equation competitive, uncompetitive and non-competitive inhibition. Competitive inhibition occurs when the inhibitor directly competes with the substrate in forming the enzyme complex. Uncompetitive inhibition involves the interaction of the inhibitor with only the enzyme-substrate complex, while non-competitive inhibition occurs when the inhibitor binds to either the enzyme or the enzyme-substrate complex without affecting the binding of the substrate. The kinetic modifications of the Michaelis-Menten equation associated with the various types of inhibition are shown below. The derivation of these equations is shown in Appendix S.S. 

The determination of kinetic mechanisms requires more than just initial velocity patterns, and inhibition studies are usually required. Several types of inhibitors are useful. The products are substrates in the reverse reaction and thus have some affinity for the enzyme and will give inhibition unless their inhibition constants exceed their solubility. Dead-end inhibitors are molecules that play musical chairs with the substrates for open portions of the active site but do not react. Substrates may act as dead-end inhibitors by combining at points in the mechanism where they are not intended and thus cause substrate inhibition. The inhibition patterns caused by these inhibitors are useful in distinguishing between different kinetic mechanisms. 

Other catalysts with firmly bound prosthetic groups, including the iron-porphyrin-bearing cytochromes, have been shown to undergo reversible oxidation. Therefore, it would form a consistent pattern if the hydroperoxidases were also to be oxidized and reduced. To explain the action of these enzymes several schemes have been advanced in which the iron shifts from the ferric to the ferrous state and back. All of these schemes have been criticized when applied to catalase because of the inability to detect a ferrous enzyme by magnetic measurements or by inhibition of the reaction with CO. It does not seem that these objections are overwhelming, as the ferrous state may be very short-lived, and escape detection by physical means, and the reduced enzyme may have less affinity for CO than those enzymes that are inhibited by this compound. 

Studies of the kinetics of the purified erythrocyte enzyme showed a highly selective product inhibition by UDP-glucose. Distinct differences were observed in the rate patterns between the forward and reverse reactions the reverse reaction showed a constant reaction rate, whereas the rate of the forward reaction, i.e., in the direction of UDP-glucose synthesis, rapidly decreased. This was the result of inhibition by the UDP-glucose formed, and further kinetic analysis showed that there was competition between UDP-glucose and UTP for free enzyme. 

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