Reversible enzyme reaction, active site

Figure 6. Enzymes act as recycling catalysts in biochemical reactions. A substrate molecule binds (reversible) to the active site of an enzyme, forming an enzyme substrate complex. Upon binding, a series of conformational changes is induced that strengthens the binding (corresponding to the induced fit model of Koshland [148]) and leads to the formation of an enzyme product complex. To complete the cycle, the product is released, allowing the enzyme to bind further substrate molecules. (Adapted from Ref. 1). See color insert.

Affinity labeling agents are intrinsically reactive compounds that initially bind reversibly to the active site of the enzyme then undergo chemical reaction (generally an acylation or alkylation reaction) with a nucleophile on the enzyme (Scheme 8). To differentiate a reversible inhibitor from an irreversible one, often the dissociation constant is written with a capital i, K (65), instead of a small i, K, which is used for reversible inhibitors. The K denotes the concentration of an inactivator that produces half-maximal inactivation. Note that this kinetic Scheme is similar to that for substrate turnover except instead of the catalytic rate constant, kcat for product formation, kmact is used to denote the maximal rate constant for inactivation. 

Specific small molecules or ions can inhibit even nonallosteric enzymes. In irreversible inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. Covalent inhibitors provide a means of mapping the enzyme s active site. In contrast, reversible inhibition is characterized by a rapid equilibrium between enzyme and inhibitor. A competitive inhibitor prevents the substrate from binding to the active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to substrate. In noncompetitive inhibition, the inhibitor decreases the turnover number. Competitive inhibition can be distinguished from noncompetitive inhibition by determining whether the inhibition can be overcome by raising the substrate concentration. 

In competitive inhibition the inhibitor, I, binds reversibly to the active site of the enzyme. Consequently, the inhibitor competes with the substrate for the active site. As more substrate is added, at constant inhibitor concentration, the inhibitor is displaced and the reaction rate approaches the same maximum value as in the absence of inhibitor. However, more substrate is required to achieve any given reaction velocity in the presence of inhibitor than in its absence. The amount of inhibition depends on the inhibitor constant, Kl, which is also the dissociation constant for the binding of the inhibitor to the enzyme ... 

There have been many attempts to explain the bell-shaped curve of enzyme activity versus Wo. It is likely that several factors contribute and that the relative importance of different parameters varies with the type of enzyme studied [40,41]. However, it seems probable that diffusion effects play a major role, and a diffusion model applicable to a hydrophilic enzyme located in the core of the water droplet and hydrophilic substrates also situated in the droplets was worked out by Walde and coworkers [42,43]. Before the enzyme-catalyzed reaction can take place, two different diffusion processes must occur. In the first of these, an interdroplet diffusion step, drops containing the substrate and drops containing the enzyme must collide. In the second process, an intradroplet diffusion step, the substrate reaches the enzyme s active site. Whereas the rate of the first process increases with droplet radius, the reverse is true for the second process. These two counteracting dependencies of reaction rate on droplet size (and thus on Wo at constant surfactant concentration) may lead to a bell-shaped activity versus Wo curve. 

There are many compounds that affect enzymatic reaction rates due to different mechanisms. Activators, such as cofactors and coenzymes, are compounds that bind with the enzyme and increase reaction rates. On the other hand, inhibitors are compounds that bind to the active site and reduce the rate by negatively influencing the catalytic properties of the enzyme s active sites (Panesar et al., 2010). In addition, an inhibitor can also bind at sites other than the enzyme s active site. For example, on the reverse, resnlting in conformational changes in the active site and a decrease in catalytic activity. 

Enzyme and substrate first reversibly combine to give an enzyme-substrate (ES) complex. Chemical processes then occur in a second step with a rate constant called kcat, or the turnover number, which is the maximum number of substrate molecules converted to product per active site of the enzyme per unit time. The kcat is, therefore, a rate constant that refers to the properties and reactions of the ES complex. For simple reactions kcat is the rate constant for the chemical conversion of the ES complex to free enzyme and products. 

The reversibility of halohydrin dehalogenase-catalyzed reactions has been used for the regioselective epoxide-opening with nonnatural nucleophiles (an example is given in Scheme 10.34) [133]. The stereoselectivity of the enzyme results in the resolution of the racemic substrate. At the same time, the regioselectivity imposed by the active site geometry yields the anti-Markovnikov product. [128]... 

The first step, which is called the acylation reaction, involves a formation of an acyl-enzyme where the RC(0 )X group is covalently bound to the specially active serine residue and the XH group is expelled from the active site. The second step, which is called the deacylation step, involves an attack of an HY group on the acyl-enzyme. Here we concentrate on the acylation step which is the reverse of the second step when X and Y are identical. 

The equilibrium constant for the second-order attachment of a substrate to the active site of an enzyme was found to be 326 at 310 K. At the same temperature, the rate constant for the second-order attachment is 7.4 X 107 L-mol-s. What is the rare constant for the loss of unreacted substrate from the active site (the reverse of the attachment reaction) ... 

The inactivation is normally a first-order process, provided that the inhibitor is in large excess over the enzyme and is not depleted by spontaneous or enzyme-catalyzed side-reactions. The observed rate-constant for loss of activity in the presence of inhibitor at concentration [I] follows Michaelis-Menten kinetics and is given by kj(obs) = ki(max) [I]/(Ki + [1]), where Kj is the dissociation constant of an initially formed, non-covalent, enzyme-inhibitor complex which is converted into the covalent reaction product with the rate constant kj(max). For rapidly reacting inhibitors, it may not be possible to work at inhibitor concentrations near Kj. In this case, only the second-order rate-constant kj(max)/Kj can be obtained from the experiment. Evidence for a reaction of the inhibitor at the active site can be obtained from protection experiments with substrate [S] or a reversible, competitive inhibitor [I(rev)]. In the presence of these compounds, the inactivation rate Kj(obs) should be diminished by an increase of Kj by the factor (1 + [S]/K, ) or (1 + [I(rev)]/I (rev)). From the dependence of kj(obs) on the inhibitor concentration [I] in the presence of a protecting agent, it may sometimes be possible to determine Kj for inhibitors that react too rapidly in the accessible range of concentration. ... 

All enzymatic reactions are initiated by formation of a binary encounter complex between the enzyme and its substrate molecule (or one of its substrate molecules in the case of multiple substrate reactions see Section 2.6 below). Formation of this encounter complex is almost always driven by noncovalent interactions between the enzyme active site and the substrate. Hence the reaction represents a reversible equilibrium that can be described by a pseudo-first-order association rate constant (kon) and a first-order dissociation rate constant (kM) (see Appendix 1 for a refresher on biochemical reaction kinetics) ... 

In this chapter we have seen that enzymatic catalysis is initiated by the reversible interactions of a substrate molecule with the active site of the enzyme to form a non-covalent binary complex. The chemical transformation of the substrate to the product molecule occurs within the context of the enzyme active site subsequent to initial complex formation. We saw that the enormous rate enhancements for enzyme-catalyzed reactions are the result of specific mechanisms that enzymes use to achieve large reductions in the energy of activation associated with attainment of the reaction transition state structure. Stabilization of the reaction transition state in the context of the enzymatic reaction is the key contributor to both enzymatic rate enhancement and substrate specificity. We described several chemical strategies by which enzymes achieve this transition state stabilization. We also saw in this chapter that enzyme reactions are most commonly studied by following the kinetics of these reactions under steady state conditions. We defined three kinetic constants—kai KM, and kcJKM—that can be used to define the efficiency of enzymatic catalysis, and each reports on different portions of the enzymatic reaction pathway. Perturbations... 

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