Allosteric enzymes, inhibitor activator effect

For example, experimental data might reveal that a novel enzyme inhibitor causes a concentration-dependent increase in Km, with no effect on and with Lineweaver-Burk plots indicative of competitive inhibition. Flowever, even at very high inhibitor concentrations and very low substrate concentrations, it is observed that the degree of inhibition levels off when some 60% of activity still remains. Furthermore, it has been confirmed that only one enzyme is present, and all appropriate blank rates have been accounted for. It is clear that full competitive inhibition cannot account for such observations because complete inhibition can be attained at infinitely high concentrations of a full competitive inhibitor. Thus, it is likely that the inhibitor binds to the enzyme at an allosteric site. 

Mechanistically, inhibition must not necessarily block the active site itself, but it can exert allosteric effects on the substrate-binding pocket, which thereby enhances or suppresses enzymatic activity. Additional considerations regarding enzymatic reactions are discussed in Reference 86. SAR by NMR has been successfully applied to various systems [i.e., for disrupting intracellular protein-protein binding (87) as well as cytokine-receptor interaction (88)]. High-affinity enzyme inhibitors have been developed by this technique [e.g., for the metalloproteinase Stromelysin (89) and the protein tyrosine phosphatase IB (90)]. 

Figure 2-18. Effect of activators and inhibitors on an allosteric enzyme.

In the concerted model, the effects of inhibitors and activators can also be considered in terms of shifting the equilibrium between the T and R forms of the enzyme. The binding of inhibitors to allosteric enzymes is cooperative allosteric inhibitors bind to and stabilize the T form of the enzyme. The binding of activators to allosteric enzymes is also cooperative allosteric activators bind to and stabilize the R form of the enzyme. When an activator, A, is present, the cooperative binding of A shifts the equilibrium between the T and R forms, with the R form favored (Figure 7.6). As a result, there is less need for substrate, S, to shift the equilibrium in favor of the R form, and less cooperativity in the binding of S is seen. 

An excellent example of allosteric regulation—the control of an allosteric enzyme—is the five-step synthesis of the amino acid isoleucine (see I Figure 10.13). Threonine deaminase, the enzyme that catalyzes the first step in the conversion of threonine to isoleucine, is subject to inhibition by the final product, isoleucine. The structures of isoleucine and threonine are quite different, so isoleucine is not a competitive inhibitor. Also, the site to which isoleucine binds to the enzyme is different from the enzyme active site that binds to threonine. This second site, called the allosteric site, specifically recognizes isoleucine, whose presence there induces a change in the conformation of the enzyme such that threonine binds poorly to the active site. Thus, isoleucine exerts an inhibiting effect on the enzyme activity. As a result, the reaction slows as the concentration of isoleucine increases, and no excess isoleucine is produced. When the concentration of isoleucine falls to a low enough level, the enzyme becomes more active, and more isoleucine is synthesized. This type of allosteric regulation in which the enzyme that catalyzes the first step of a series of reactions is inhibited by the final product is called feedback inhibition. 

One of the most used and effective methods to inhibit enzymes using organometallic complexes, and more generally metal complexes, is by covalent coordination of a metal center to a residue of a specific amino acid involved in the catalytic activity of the enzyme (at active or allosteric sites). This metal coordination renders the enzyme inactive, usually in an irreversible manner (see Scheme 1.2 for an example with a tyrosine phosphatase and a gold organometallic complex inhibitor [51]). More specifically, the catalytic mechanism of tyrosine phosphatases relies on the transfer of a phosphate group from the substrate to a cysteine residue in the catalytic site (Scheme 1.2, path (a)). The covalent coordination of the gold center to the cysteine residue allows for the... 

Activators and inhibitors regulate not the amount of enzyme protein but the activity ( efficiency ) of that which is present. Two principal mechanisms of control are (i) competitive and (ii) allosteric. Competitive control (inhibition) occurs when a compound which is structurally similar to the true substrate binds to the active site of the enzyme. This is how a number of drugs and poisons bring about their effect. For example, a group of therapeutic drugs called statins are used to treat heart disease because by inhibiting a key enzyme called HMGCoA reductase, they reduce the hepatic synthesis of cholesterol and therefore the plasma concentration of that lipid. 

The fact that ATP and CTP bind to the same site follows from the observation that adding ATP to the inhibited enzyme by CTP reduces or reverses the inhibition, presumably because ATP competes with CTP for the same site. The fact that CTP binds to an allosteric site (i.e., it is not a competitive inhibitor) follows from the so-called desensitization effect. Addition of mercurials [e.g., p-mercuribenzoate (PMB)] reduces or eliminates the inhibition by CTP. However, it has no effect on the enzymatic activity of ATCase, presumably because the mercurials affect the regulatory subunits but not the catalytic site. As for the mechanism of cooperativity (both positive and negative), it is known that CTP does induce changes in the quaternary structure of the enzyme. 

Allosteric inhibitors bind to a separate binding site outside the active center (6). This results in a conformational change in the enzyme protein that indirectly reduces its activity (see p. 116). Allosteric effects practically only occur in oligomeric enzymes. The kinetics of this type of system can no longer be described using the simple Micha-elis-Menten model. 

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