Reversible inhibition patterns

Cleland described the following rules for reversible inhibition patterns observed in double-reciprocal plots of initial rate behavior. 

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. 

Competitive inhibition results when an inhibitor, I, binds reversibly to the active site and prevents the substrate, S, from binding, and vice versa. Both I and S are mutually exclusive on the active site. The equilibria describing this inhibition pattern are shown in Eqn. 7.25. 

The statement that reversible inhibition mechanisms only exhibit ray patterns seems to contradict experience. Often, the experimentally determined B ax seems to decrease with increasing L, even when the measured values are correct and the mechanism is reversible. The root cause of this phenomenon is nonlinear Scatchard plots. For the experimenter, who cannot measure all points of the curve, they create the illusion of a decreasing B ax (Rehm and Becker 1988). For inhibition mechanism I (competitive), the inhibitor bends the Scatchard plots downward, and the commonly performed interpolation of the measuring points creates the appearance of decreasing B , with increasing L (see Figure 2.23). 

In these experiments we have studied the degradation of the D1 protein in Cl-depleted thylakoid membranes as a model for the situation in non-depleted thylakoid membranes. We have chosen this material since our spectroscopic ex riments suggests that the degradation of the D1 protein is triggered from reactions on the oxiSzing side of PSII despite the fact that the inhibition of the electron tranfer is at the level of die function of Qa inhibition pattern in Cl-depleted thylakoid membranes is well established and is known to result from a block in the S-cycle between the S2 and the S3 state. Furthermore, the inhibition induced by Cl-depletion is reversible which shows that this material is less modified than for example NH2OH- or TRIS-washed thylakoids which also are known to be inhibited on die donor side of PSII. 

The hydroxylated derivatives, 5-hydroxy-2-formylpyridine thiosemicarbazone and 3-hydroxy-2-formylpyridine thiosemicarbazone, show a different pattern of inhibition [74]. They appear competitive with iron and either non-competitive or uncompetitive with the dithiol substrate the imprecise nature of the assay does not allow a choice between these alternatives. The failure of the dithiol to reverse inhibition of ribonucleoside diphosphate reductase by the hydroxylated derivatives implies that the interaction of these inhibitors with the enzyme occurs at a site different from that involved in the action of (4) and (1). The impure nature of this complex enzyme system, however, makes it impossible to explain fully these differences and further advances will require the availability of a highly purified enzyme. 

Inhibition of methyl transferase by 5-azacytidine (Fig. 1.42) leads to a change in the methylation pattern and to a reversal of differentiation of a cell culture. 

Diethanolamine has been shown to inhibit choline uptake into cultured Syrian hamster embryo (SHE) and Chinese hamster ovary cells and to inhibit the synthesis of phosphatidylcholine in in-vitro systems in a concentration-dependent, competitive and reversible manner (Lehman-McKeeman Gamsky, 1999, 2000). Diethanolamine treatment caused a marked reduction in hepatic choline metabolite concentrations in mice following two weeks of dermal dosing. The most pronounced reduction was in the hepatic concentration of phosphocholine, the intracellular storage form of choline (Stott et al, 2000). Moreover, the pattern by which choline metabolites were altered was similar to the pattern of change that has been observed following dietary choline deprivation in rodents (Pomfret et al, 1990). Excess choline also prevented diethanolamine-induced inhibition of phosphatidylcholine synthesis and incorporation of diethanolamine into SHE cell phospholipids (Lehman-McKeeman Gamsky, 2000). 

A fourth pattern of interaction (enzymes of group D) between allosteric activator and inhibitor is seen with barley endosperm. The ADPGIc PPase, which is poorly-activated by 3PGA, is inhibited by Pi (Table 4.2). However, 3PGA lowers (up to 3-fold) the S0 5 for ATP (i.e. the apparent affinity of ATP is increased) and the Hill coefficient.75 At 0.1 mM ATP, activation by 3PGA is about 4-fold 2.5 mM Pi reverses the effect. Thus, in barley endosperm, the prime effect of 3PGA or Pi may be to either increase or decrease the apparent affinity of the enzyme for the substrate, ATP. 

The very heterogeneous chemical structure of the compounds with MDR reversal activity has prevented structure-activity studies, although most MDR-inhibiting molecules share a basic structural pattern comprising a cationic protonable site linked to an aromatic lipophilic part by a spacer of variable length [61]. Structure-activity relationship (SAR) studies yielded only qualitative indications [62-64] unless very homogeneous series of molecules are studied [65]. 

In noncompetitive inhibition, which also is reversible, the inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites (see Figure 8.16). A noncompetitive inhibitor acts by decreasing the turnover number rather than by diminishing the proportion of enzyme molecules that are bound to substrate. Noncompetitive inhibition, in contrast with competitive inhibition, cannot be overcome by increasing the substrate concentration. A more complex pattern, called mixed inhibition, is produced when a single inhibitor both hinders the binding of substrate and decreases the turnover number of the enzyme. 

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