Inhibitor binding reaction mechanism

In this chapter we described the thermodynamics of enzyme-inhibitor interactions and defined three potential modes of reversible binding of inhibitors to enzyme molecules. Competitive inhibitors bind to the free enzyme form in direct competition with substrate molecules. Noncompetitive inhibitors bind to both the free enzyme and to the ES complex or subsequent enzyme forms that are populated during catalysis. Uncompetitive inhibitors bind exclusively to the ES complex or to subsequent enzyme forms. We saw that one can distinguish among these inhibition modes by their effects on the apparent values of the steady state kinetic parameters Umax, Km, and VmdX/KM. We further saw that for bisubstrate reactions, the inhibition modality depends on the reaction mechanism used by the enzyme. Finally, we described how one may use the dissociation constant for inhibition (Kh o.K or both) to best evaluate the relative affinity of different inhibitors for ones target enzyme, and thus drive compound optimization through medicinal chemistry efforts. 

Bimolecular processes are very common in biological systems. The binding of a hormone to a receptor is a bimolecular reaction, as is substrate and inhibitor binding to an enzyme. The term bimolecular mechanism applies to those reactions having a rate-limiting step that is bimolecular. See Chemical Kinetics Molecularity Reaction Order Elementary Reaction Transition-State Theory... 

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. 

Suicide inhibitors, alternatively known as Kcat or irreversible mechanism based inhibitors (IMBIs), are irreversible inhibitors that are often analogues of the normal substrate of the enzyme. The inhibitor binds to the active site, where it is modified by the enzyme to produce a reactive group, which reacts irreversibly to form a stable inhibitor-enzyme complex. This subsequent reaction may or may not involve functional groups at the active site. This means that suicide inhibitors are likely to be specific in their action, since they can only be activated by a particular enzyme. This specificity means that drugs designed as suicide inhibitors could exhibit a lower degree of toxicity. 

An inhibitor is any agent that interferes with the activity of an enzyme. Inhibitors may affect the binding of enzyme to substrate, or catalysis (via modification of the enzyme s active site), or both. Researchers use enzyme inhibitors to define metabolic pathways and to understand enzyme reaction mechanisms. Many drugs are designed as inhibitors of target enzymes. Inhibition is also a natural phenomenon. Cells regulate metabolic pathways by specific inhibition of key enzymes. 

The similarities concerning the bound cofactors, the reaction mechanism, and the adenine-binding pockets led to a screen of the above-described purine-based library of ATP-competitive inhibitors originally designed to target CDKs for crossreactivity with the carbohydrate sulfotransferase NodH from Rhizobium meliloti. 

The reaction mechanism of cytochrome bc complex is known as the proton motive Qcycle originally proposed by Peter Mitchell (Mitchell, 1976). This mechanism is the basis of his chemiosmotic theory for which he was awarded the Nobel prize in 1978. Since then, the enzyme has been characterized extensively using various techniques. Redox centers have been characterized spectroscopically (for review, see Trumpower and Gennis, 1994), electron transfer pathways have been determined using kinetic experiments with specific inhibitors (De Vries 1986 Zhu et al., 1984), and the positions of quinone binding sites and redox centers have been determined using biochemical and mutational analysis (for review, see Esposti et al, 1993 Brasseur et al, 1996). As a result of these efforts, the latest modified Qcycle has been widely accepted by researchers in the field (for reviews, see Crofts et al, 1983 Trumpower, 1990 Berry et al, 2000). 

COMT is, for many of the same reasons as with chorismate mutase, well suited for the study with computational techniques. The reaction mechanism it catalyzes is the same mechanism that operates in the absence of the enzyme, specifically, the S 2 mechanism, facilitating comparison of the bare solution-phase reaction with the catalyzed reaction. The subsfiate and cofactor do not covalendy bind to the enzyme, so that defining the QM region and the MM region should be relatively uncomplicated. Lasdy, the X-ray crystal structure of COMT bound with the inhibitor 3,5-dinitrocatechol has been determined with a resolution of 2 kP An interesting twist to this enzyme is that the active site includes a metal cation, Mg " ". This crystal structure allows for a natural starting point for computational exploration of the means of the catalytic action of COMT. The rate acceleration provided by COMT is substantial the reaction is 10 times faster within the enzyme than in solution. " ... 

Most commercial HPPD inhibitors e.g., sulcotrione and isoxaflutole) are competitive time-dependent (tight-binding) inhibitors. As such, these herbicides bind to the enzyme very tightly with T A of dissociation ranging from a few hoins to several days, as opposed to milliseconds for traditional reversible inhibitors. Sorgoleone does not behave as these herbicides and appears to be a reversible inhibitor of HPPD. This quinone is structurally more planar than the traditional HPPD inhibitors, so it may not form a stable reaction intermediate. Instead, its backbone may resemble the conformation of one of the later intermediate step in the reaction mechanism of HPPD. 

Oximes bind to AChE as reversible inhibitors and form complexes with AChE either at the acylation (catalytic) site, at the allosteric site, or at both sites of the enzyme and protect AChE from phosphorylation. When the reversible inhibitor binds to the catalytic site, the protection is due to direct competition between OP and reversible inhibitor. Binding of a reversible inhibitor to the allosteric site induces indirect protection of the active site. Differences in the mechanisms of enzyme reactivation and protection demonstrate how stereochemical arrangements of oximes can play a role in the potency of their therapeutic efficacy. Direct pharmacological effects, such as direct reaction with OPs (Van Helden et al., 1996), anticholinergic and sympathomimetic effects may also be relevant for the interpretation of antidotal potency of oximes. 

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