Initial Binding of Substrate

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)  

Evaluation of Enzyme Inhibitors in Drug Discovery, by Robert A. Copeland ISBN 0-471-68696-4 Copyright 2005 by John Wiley Sons, Inc. 

The binary complex ES is commonly referred to as the ES complex, the initial encounter complex, or the Michaelis complex. As described above, formation of the ES complex represents a thermodynamic equilibrium, and is hence quantifiable in terms of an equilibrium dissociation constant, Kd, or in the specific case of an enzyme-substrate complex, Ks, which is defined as the ratio of reactant and product concentrations, and also by the ratio of the rate constants kM and km (see Appendix 2)  

The equilibrium dissociation constant Ks has units of molarity and its value is inversely proportional to the affinity of the substrate for the enzyme (i.e., the lower the value of Ks, the higher the affinity). The value of Ks can be readily converted to a thermodynamic free energy value by the use of the familiar Gibbs free energy equation  

as described by Equation (2.1), the equilibrium dissociation constant depends on the rate of encounter between the enzyme and substrate and on the rate of dissociation of the binary ES complex. Table 2.1 illustrates how the combination of these two rate constants can influence the overall value of Kd (in general) for any equilibrium binding process. One may think that association between the enzyme and substrate (or other ligands) is exclusively rate-limited by diffusion. However, as described further in Chapter 6, this is not always the case. Sometimes conformational adjustments of the enzyme s active site must occur prior to productive ligand binding, and these conformational adjustments may occur on a time scale slower that diffusion. Likewise the rate of dissociation of the ES complex back to the free 

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By changing Ser 221 in subtilisin to Ala the reaction rate (both kcat and kcat/Km) is reduced by a factor of about 10 compared with the wild-type enzyme. The Km value and, by inference, the initial binding of substrate are essentially unchanged. This mutation prevents formation of the covalent bond with the substrate and therefore abolishes the reaction mechanism outlined in Figure 11.5. When the Ser 221 to Ala mutant is further mutated by changes of His 64 to Ala or Asp 32 to Ala or both, as expected there is no effect on the catalytic reaction rate, since the reaction mechanism that involves the catalytic triad is no longer in operation. However, the enzyme still has an appreciable catalytic effect peptide hydrolysis is still about 10 -10 times the nonenzymatic rate. Whatever the reaction mechanism... 

Conceptually, carrier-mediated transport across a membrane involves 3 steps (Anwer, 1976), 1) Initial binding of substrate to carrier at the outer membrane surface 2) Rotation or translation of carrier-substrate complex within the membrane and 3) Dissociation of carrier-substrate complex at inner membrane surface. Thus, K[ or the rate constant of translocation involves more than the affinity of a substrate(s) for its carrier and is in fact inversely proportional to the overall efficiency of carrier-mediated transport of a given compound across a membrane. 

When an appropriate chiral phosphine ligand and proper reaction conditions are chosen, high enantioselectivity is achievable. If a diphosphine ligand with C2 symmetry is used, two diastereomers for the enamide-coordinated complex can be formed because the olefin can interact with the metal from either the Re- or Sf-face. Therefore, enantioselectivity is determined by the relative concentrations and reactivities of the diastereomeric substrate-Rh complexes. It should be mentioned that in most cases it is not the preferred mode of initial binding of the prochiral olefinic substrate to the catalyst that dictates the final stereoselectivity of these catalyst systems. The determining factor is the differ-... 

Knowing the initial concentrations of substrate and ligand and the fraction of unbound substrate in the reaction mixture, the association constant can be calculated. The binding isotherm needs the measurement of five to ten reaction mixtures of different initial concentration ratios, but with commercial instruments this is easily automated, and the higher consumption of sample volume (about 80 nL) doesn t matter. 

Initial binding of all substrates is followed by an isomerization or conformational change with a characteristic time constant of the order of magnitude of 103 sec-1. 

Supramolecular photochemistry, like catalysis, may involve three steps binding of substrate and receptor, mediating a photochemical process (such as energy, electron or proton transfer), followed by either restoration of the initial state for a new cycle or by a chemical reaction (Figure 16). 

The conversion of a substrate S into product P by an enzyme involves initial binding of the substrate to the enzyme and subsequent breakdown of the enzyme-substrate complex into product. In the simplest scheme for a single substrate-single product... 

Kinetic studies by Hayaishi and his co-workers revealed that the enzyme catalyzed reaction proceeded with initial binding of the substrate to the enzyme and subsequent binding of 02 to the ES complex45). Under stopped flow conditions, an ES02 intermediate was observed, which decayed with a rate in agreement with the turnover number for the enzyme45). 

Inhibition of acetylcholinesterase by an organophosphate. The initial binding of the organophos-phate to the active site prevents the normal substrate from entering the active site. The aging process subsequently binds the organophosphate to the active site permanently, inactivating the enzyme. 

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