Inhibitors Bind at the Active Site

Types of enzyme inhibition, (a) A competitive inhibitor competes with the substrate for binding at the same site on the enzyme, (b) A noncompetitive inhibitor binds to a different site but blocks the conversion of the substrate to products, (c) An uncompetitive inhibitor binds only to the enzyme—substrate complex. (E = enzyme S = substrate.) 

An expression describing enzyme kinetics in the presence of a competitive inhibitor can be derived straightforwardly. Consider the reaction that we treated previously. 

The specificity pocket of trypsin can accommodate an arginine side chain of a polypeptide substrate or a benzamidine ion, which acts as a competitive inhibitor. Asp = aspartic acid. 

Competitive inhibition. A series of double-reciprocal plots (1/v versus 1/[S]) measured at different concentrations of the inhibitor (I) all intersect at the same point (on the ordinate. The slopes of the plots and the intercepts on the abscissa are simple, linear functions of [IJ/A j, where K, is the dissociation constant of the inhibitor-enzyme complex. 

We now have the additional feature that the enzyme also reacts reversibly with the inhibitor (I) to give an inactive complex (El). 

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A graphical procedure for assessing the possible interaction of two different inhibitors binding at the active site of an enzyme. In this method, the investigator plots vjvi as a function of the concentration of one of the two inhibitors at different, constant concentrations of the second substrate . Such plots provide a means for the calculation of the inhibitor interaction constant which provides the investigator with a tool for assessing the degree of overlap of subsites at an enzyme s active center. 

Several inhibitor-protease complexes have been crystallized and details of their interactions are known. For example, the pancreatic trypsin inhibitor binds at the active site of trypsin with K( >1013 M-1 at neutral pH 496 Tire two molecules fit snugly together,490 497 the inhibitor being bound as if it were a peptide substrate with one edge of the inhibitor molecule forming an antiparallel (1 structure with a peptide chain in the enzyme. Lysine 15, which forms part of this P structure, enters the specific Pj binding site for a basic amino acid in a substrate. Thus, the protease inhibitor is a modified substrate which may actually undergo attack at the active site. However, the fit between the two... 

Competitive Inhibitors Bind at the Active Site Noncompetitive and Uncompetitive Inhibitors Do Not Compete Directly with Substrate Binding Irreversible Inhibitors Permanently Alter the Enzyme Structure... 

Reversible inhibitors bind an enzyme through weak, intermolecular forces and establish an equilibrium of being bound or unbound to the enzyme. A competitive inhibitor binds at the active site and prevents the substrate from binding. A noncompetitive inhibitor binds an allosteric site on the enzyme and prevents conversion of the substrate to product. Uncompetitive inhibitors bind the enzyme-substrate complex and make it inactive. All three types of inhibitors show characteristic, distinctive features in a Lineweaver-Burk plot. 

The other extreme is when a compound binds only to the E S complex but not to the free enzyme, in which case uncompetitive inhibition occurs (Scheme 2). Although it is rare in single substrate reactions, it is common in multiple substrate systems. An inhibitor of a two-substrate enzyme that is competitive against one of the substrates often is found to give uncompetitive inhibition when the other substrate is varied. The inhibitor binds at the active site but only prevents the binding of one of the substrates. 

Figure 8.15. Distinction between a Competitive and a Noncompetitive Inhibitor. (Top) enzyme-substrate complex (middle) a competitive inhibitor binds at the active site and thus prevents the substrate from binding (bottom) a noncompetitive inhibitor does not prevent the substrate from binding.

Inhibitors are species that bind to enzymes, modifying their activity. Competitive inhibitors bind at the same site as the substrate binds this is analogous to competitive adsorption in heterogeneous catalysis. The reaction scheme becomes ... 

Various mechanisms of reversible inhibition have been proposed. Competitive inhibition is conceptually the easiest to understand. Recall that the active site of an enzyme is complementary in shape to the shape of the substrate (crudely, the lock and key hypothesis). Suppose a compound which is not the true substrate, but structurally similar to it blocks the active site by binding to it. The true substrate cannot bind and so no reaction will occur. Hence, there is competition between the true substrate and the inhibitor for binding at the active site. 

Three important classes of inhibitors are shown in Table 1-8-3. Competitive inhibitors resemble the substrate and compete for binding to the active site of the enzyme. Noncompetitive inhibitors do not bind at the active site. They bind to regulatory sites on the enzyme. Irreversible inhibitors inactivate the enzyme similar to removing enzyme from the assay. 

Fig. 3. Structure of the inhibitor phosphoramidon. The tetrahedral phosphorus atom binds at the active site of thermolysin and mimics the transition-state complex.

An experiment with an irreversible inhibitor should carry with it a control experiment involving the addition of a substrate if the location of the reaction with inhibitor is at the active site, then the addition of a substrate will slow down the rate of inhibition. For example, the reactivity of papain (5 pM) with a 1.71 pM solution of 4-toluenesulphonylamidomethyl chloromethyl ketone suffers a drop of 1.68-fold when the substrate (methyl hippurate) is changed from 12.7 to 21.1 mM. The inhibitor which reacts covalently with the enzyme should carry either a radioactive or spectroscopic tag which would enable the location of the altered amino acid to be determined in the sequence, and hence in the three-dimensional X-ray crystallographic map of the enzyme. An alternative approach is to design an inhibitor with groups (analogous to those attached to the substrate) which force it to bind at the active site (Scheme 11.18). 

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. 

Competitive inhibition occurs, when substrate and inhibitor compete for binding at the same active site at the enzyme. Based on the Michaelis-Menten kinetics, Vmax is unchanged whereas Km increases. In case of noncompetive inhibition, the inhibitor and the substrate bind to different sites at the enzyme. Vmax decrease whereas the Km value is unaffected. Binding of the inhibitor only to the enzyme-substrate complex is described as uncompetitive inhibition. Both, Vmax and Km decrease. Finally, mixed (competitive-noncompetitive) inhibition occurs, either the inhibitor binds to the active or to another site on the enzyme, or the inhibitor binds to the active site but does not block the binding of the substrate. 

There are many other questions that need to be addressed. For example What are the kinetics of the inhibition Do the different inhibitors bind at the same site What are the molecular requirements for inhibition What are the differences between susceptible and tolerant ACCases and so on. ACCase purified 40 to 100 fold may not be sufficiently pure to answer many of these questions. For example, an extract purified on a Sephacryl S-300 column can have a specific activity up to 400 nmol/min/mg. We have observed that this preparation can catalyze the carboxylation of other short chained acyl CoA s in addition to acetyl CoA (Table VI). Both haloxyfop and tralkoxydim inhibit the carboxylation reaction regardless of whether n-propionyl CoA or acetyl CoA are substrates either individually or together (Table VII). At present, we are unsure whether n-propionyl CoA can be used as a substrate for ACCase or whether a n-propionyl CoA carboxylase is present in the preparation and the herbicides also inhibit that enzyme. 

Reversible inhibition can be competitive or non-competitive. Competitive inhibitors bind to the active site and compete with the substrate for binding to the enzyme. However this means that increasing the S concentration will progressively outcompete the inhibitor. Accordingly a Lineweaver—Burk analysis of enzyme kinetic data obtained in the presence or absence of a competitive inhibitor will yield the same Fmax (at infinite S concentration) but the Am in the presence of the inhibitor (A, ) will be higher (poorer binding) than the Am measured in the absence of competitive inhibitor. Knowing the inhibitor concentration [I] one can calculate the A) from the relation ... 

Animal proteases, particularly those involved in blood clotting, can be also regulated by endogenous protease inhibitory proteins that act as inhibitory substrate analogues. These inhibitor proteins bind at the active site through key inhibitory sequences in which the key residues about the peptide bond contribute to inhibition and are denoted (N-terminal side)—P2—PI—(peptide bond to be hydrolysed)-PT-P2 -(C-terminal side) or simply P2-P1-PT-P2. A large number of plant PI proteins also act as peptide substrate mimetics. 

Bioenergetics provides a quantitative description of the transformation of materials and energy in living systems. Most biochemical reactions occur in pathways, in which other reactions continuously add substrates and remove products. The rate of reactions depends on the properties of the enzymes (large proteins produced in cells) that catalyze the reaction. Substrates bind at the active sites of enzymes, where they are converted to products and later released. Enzymes are highly specific for given substrates and products. Inhibitors of enzymes decrease the rate of reaction. 

Most irreversible enzyme inhibitors combine covalently with functional groups at the active sites of enzymes. These inhibitors are usually chemically reactive, and many of them show some specificity in terms of the amino acid groups which they react with. Diisopropyl fluorophosphate (DFP), for example, forms a covalent adduct with active site serine residues, such as in the serine proteases, and in acetylcholinesterase, which explains its toxic effect on animals. Irreversible enzyme inhibition can be used to identify important active site residues. A special case of irreversible enzyme inhibition is the effect of suicide inhibitors, which are generally chemically unreactive compounds that resemble the substrate of the target enzyme and bind at the active site. The process of enzyme turnover begins, but the inhibitor is so... 

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