Allosteric enzymes, inhibitor

To refer to the kinetics of allosteric inhibition as competitive or noncompetitive with substrate carries misleading mechanistic implications. We refer instead to two classes of regulated enzymes K-series and V-se-ries enzymes. For K-series allosteric enzymes, the substrate saturation kinetics are competitive in the sense that is raised without an effect on V. For V-series allosteric enzymes, the allosteric inhibitor lowers... 

In a very broad overview of the structural categories one can state several statistical correlations with type of function. Hemes are almost always bound by helices, but never in parallel a//3 structures. Relatively complex enzymatic functions, especially those involving allosteric control, are occasionally antiparallel /3 but most often parallel a//3. Binding and receptor proteins are most often antiparallel /3, while the proteins that bind in those receptor sites (i.e., hormones, toxins, and enzyme inhibitors) are most apt to be small disulfide-rich structures. However, there are exceptions to all of the above generalizations (such as cytochrome cs as a nonhelical heme protein or citrate synthase as a helical enzyme), and when one focuses on the really significant level of detail within the active site then the correlation with overall tertiary structure disappears altogether. For almost all of the dozen identifiable groups of functionally similar proteins that are represented by at least two known protein structures, there are at least... 

Enzymatic reactions can be impeded by the addition of exogenous molecules. This is how drugs are used to control biochemical reactions, and most drugs are used for inhibitory functions. Drugs may function as competitive inhibitors or as noncompetitive inhibitors. Competitive inhibitors compete with the substrates for binding to the active sites, whereas noncompetitive inhibitors bind to another location (allosteric site) but affect the active site and its consequential interactions with the substrates. Some drugs used as enzyme inhibitors are the following ... 

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. 

Most enzyme inhibitors act reversibly—i. e., they do not cause any permanent changes in the enzyme. However, there are also irreversible inhibitors that permanently modify the target enzyme. The mechanism of action of an inhibitor—its inhibition type—can be determined by comparing the kinetics (see p.92) of the inhibited and uninhibited reactions (B). This makes it possible to distinguish competitive inhibitors (left) from noncompetitive inhibitors (right), for example. Allosteric inhibition is particularly important for metabolic regulation (see below). 

In addition to being easier to fit than the hyperbolic Michaelis-Menten equation, Lineweaver-Burk graphs clearly show differences between types of enzyme inhibitors. This will be discussed in Section 4.5. However, Lineweaver-Burk equations have their own distinct issues. Nonlinear data, possibly indicating cooperative multiunit enzymes or allosteric effects, often seem nearly linear when graphed according to a Lineweaver-Burk equation. Said another way, the Lineweaver-Burk equation forces nonlinear data into a linear relationship. Variations of the Lineweaver-Burk equation that are not double reciprocal relationships include the Eadie-Hofstee equation7 (V vs. V7[S]) (Equation 4.14) and the Hanes-Woolf equation8 ([S]/V vs. [S]) (Equation 4.15). Both are... 

NRTIs, which act as a substrate for RT, are not the only means of inhibiting reverse transcriptase. Like any enzyme, inhibitors can also bind allosteric positions away from the active site. An allosteric site on RT has been successfully targeted by drugs, and these drugs are called non-nucleoside reverse transcriptase inhibitors (NNRTIs). The three most frequently prescribed NNRTIs are shown in Figure A.46. 

Catalyst inhibition is traditionally associated with biocatalytic processes, but can also apply to homogeneous and heterogeneous catalysis. Competitive inhibition is analogous to competitive adsorption in gas/solid heterogeneous catalysis, where two molecules from the gas phase compete for the same active site on the catalyst surface. A competitive inhibitor is any chemical species I which can bind to the same site as the substrate, or to another site on the enzyme (an allosteric site). The overall reaction scheme is then given by Eqs. (2.58)-(2.60), where El indicates an enzyme-inhibitor complex. 

It should be noted that the mechanism depicted in Scheme 1 is the simplest that is consistent with mechanism-based inhibition. The mechanism for a given inhibitor and enzyme may be considerably more complex due to (a) multiple intermediates [e.g., MIC formation often involves four or more intermediates (29)], (b) detectable metabolite that may be produced from more than one intermediate, and (c) the fact that enzyme-inhibitor complex may produce a metabolite that is mechanistically unrelated to the inactivation pathway. Events such as these will necessitate alternate definitions for Z inact, Kh and r in terms of the microrate constants of the appropriate model. The hyperbolic relationship between rate of inactivation and inhibitor concentration will, however, remain, unless nonhyperbolic kinetics characterize this interaction. Silverman discussed this possibility from the perspective of an allosteric interaction between inhibitor and enzyme (5). Nonhyperbolic kinetics has been observed for the interaction of several drugs with members of the CYPs (30). 

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)]. 

It should be noted that we will concentrate on inhibitors directed at the active site of the enzyme. While recognizing that there are inhibitors that bind to regions other than the active site, such as allosteric effectors, these are not the focus of this chapter and will not be included. There are many reviews of enzyme inhibitors available in the literature (37, 46-48) and the reader is referred to them for more detailed analysis. 

ENZYME INHIBITORS are important in medicinal chemistry, pharmacology and therapeutics for a number of reasons. Mechanistically, they may act in a number of different ways, mainly as competitive antagonists or allosteric modifiers, and sometimes as irreversible antagonists. Many important enzyme inhibitors are within drug classes vital to everyday therapeutics. Most of the important classes are discussed in more detail elsewhere. 

A detailed characterization of the purified wheat endosperm ADP-Glc PPase has shown that the enzyme is regulated in a different manner by metabolites. The wheat endosperm enzyme is allosterically inhibited by Pi, ADP, and Fru 1,6-bisP. These inhibitions can be reversed by 3PGA and F6P. But 3PGA and F6P have no effect on enzyme activity in the absence of the inhibitors. The wheat endosperm ADP-Glc PPase thus has distinctive regulatory properties and is placed in class IX (Table 1). 

Allosteric activators and inhibitors (allosteric effectors) are compounds that bind to the allosteric site (a site separate from the catalytic site) and cause a conformational change that affects the affinity of the enzyme for the substrate. Usually an allosteric enzyme has multiple interacting subunits that can exist in active and inactive conformations, and the allosteric effector promotes or hinders conversion from one conformation to another. 

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