Substrate inhibition conformational changes

ISOTOPE TRAPPING STICKY SUBSTRATES Substrate-induced conformational change, INDUCED FIT MODEL SUBSTRATE INHIBITION ABORTIVE COMPLEX FORMATION LACTATE DEHYDROGENASE LEE-WILSON EQUATION... 

Rittinger K, Divita G, Goody R. Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors. Proc Natl Acad Sci USA 1995 92 8046-8049. 

Anti-ribosomal antibiotics are enzyme inhibitors, and the ones that are dinically useful inhibit bacterial ribosomes far more effectively than they inhibit eukaryotic ribosomes. Many enzyme inhibitors exert their effects by binding to the active site of enzymes and thereby prevent the binding of substrates. Others block enzyme function by inhibiting conformational changes essential for enzyme activity. Anti-ribosomal antibiotics are unexceptional in this regard. They bind to the sites on both subunits to which tRNA also binds. Some block the interactions of substrates with the ribosome. Others block the tunnel and thereby prevent the nascent peptide from extending. 

Unlike other enzymes that we have discussed, the completion of a catalytic cycle of primer extension does not result in release of the product (TP(n+1)) and recovery of the free enzyme. Instead, the product remains bound to the enzyme, in the form of a new template-primer complex, and this acts as a new substrate for continued primer extension. Catalysis continues in this way until the entire template sequence has been complemented. The overall rate of reaction is limited by the chemical steps composing cat these include the chemical step of phosphodiester bond formation and requisite conformational changes in the enzyme structure. Hence there are several potential mechanisms for inhibiting the reaction of HIV RT. Competitive inhibitors could be prepared that would block binding of either the dNTPs or the TP. Alternatively, noncompetitive compounds could be prepared that function to block the chemistry of bond formation, that block the required enzyme conformational transition(s) of turnover, or that alter the reaction pathway in a manner that alters the rate-limiting step of turnover. 

A noncompetitive inhibitor is one that inhibits the enzyme and its inhibitory activity is unaffected by substrate, i.e., it will inhibit the enzyme to the same degree whether the substrate is present or not. This is generally thought to occur by the inhibitor binding at some site other than the substrate-binding site but in a way that inactivates the enzyme, e.g., induced conformational change of the active site. Therefore, we may have inhibitor binding reversibly to free enzyme [Eq. (3.22)] or to the enzyme substrate complex [Eq. (3.23)], but in both cases the bound enzyme is inactive. 

Allosteric enzymes show various activation and inhibition effects which are competitive in nature and related to conformational changes in the structure of the enzyme. Such allosteric enzymes are often crucial enzymes in metabolic pathways and exert control over the whole sequence of reactions. The name allostery refers to the fact that inhibition of the enzyme is by substances that are not similar in shape to the substrate. 

To provide a mechanism for the feedback inhibition of these enzymes, the allosteric model was put forward in 1963. It was proposed that the enzyme that regulates the flux through a pathway has two distinct binding sites, the active site and a separate site to which the regulator binds. This was termed the allosteric site. The word allosteric means different shape , which in the context of this mechanism means a different shape from the substrate. The theory further proposed that when the regnlator binds to the allosteric site, it canses a conformational change in... 

Tire effects of inhibitors or activators on the kinetics of the monomeric enzyme of Fig. 9-13 can be described by Eq. 9-62 to 9-64. Separate terms for both inhibition and activation can be included. Tire equilibrium between the two conformers can also be indicated explicitly according to Eq. 7-30. However, for monomeric enzymes it is usually not profitable to try to separate the two constants Kt and KttX which describe the conformational change and binding of substrate or activator, respectively, in Eq. 7-30. 

Many inhibitors with very low dissociation constants appear to have a slow onset of inhibition when they are added to a reaction mixture of enzyme and substrate. This was once interpreted as the inhibitors having to induce a slow conformational change in the enzyme from a weak binding to a tight binding state. But in most cases, the slow binding is an inevitable consequence of the low concentrations of inhibitor used to determine its Ki. For example, consider the inhibition of trypsin by the basic pancreatic trypsin inhibitor. Kx is 6 X 10-14 M and the association rate constant is 1.1 X 106 s-1 M-1 (Table 4.1). To determine the value of Ki, inhibitor concentrations should be in the range of K1, where the observed first-order rate constant for association is (6 X Q U M) X (1.1 X 106 s-1 M-1) that is, 6.6 X 10-8 s 1. The half-life is (0.6931/6.6) X 108 s, which is more than 17 weeks. 

Uncompetitive inhibitors bind only to the enzyme-substrate complex and not to the free enzyme. For example, the substrate binds to the enzyme causing a conformational change which reveals the inhibitor binding site, or it could bind directly to the enzyme-bound substrate. In neither case does the enzyme compete for the same binding site, so the inhibition cannot be overcome by increasing the substrate concentration. Scheme 5.A5.2 below illustrates this uncompetitive behaviour. 

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