Substrate and Transition State Binding

Tutt and Schwartz (1971) studied the basic cleavage of some penicillin derivatives [31]—> [32] in the presence of /3-CD. For acyl groups bearing alkyl and aryl substituents of various sizes and geometries, the accelerations ( c/ u) vary less (31-89) than the spread of Ks values (3.85-75 mM) (Table A5.16). Approximately, therefore, the XTS values vary in parallel with Ks, suggesting that the acyl group is involved in substrate and transition state binding to about the same extent. 

Figures, Influence of mutation at Aigi27 on substrate and transition state binding to carboxypeptidase A each group of bars represents, L to R, the wild t e, R127K, K127M, and R127A mutants, and R127A mutant + 0.5 M guanidine HQ respectively.

The cleavage of /7-nitrophenyl alkanoates (222 n = 1-8) at high pH is modestly catalysed by micelles formed from cetyltrimethylammonium bromide (CTAB) in aqueous solution. Rate constants exhibit saturation behaviour with respect to [CTAB], consistent with substrate binding in the micelles. The strength of substrate binding and transition state binding to the micelles increases monotonically with the acyl chain length, and with exactly the same sensitivity. As a result, the extent of acceleration... 

Detailed binding with substrate and transition state analogs has also been reported on KSI [83, 84] using a wide range of techniques, highlighting the subtle interplay of the electrostatic and geometric properties of the enolate stabilizing active site. 

C. A. Miller, P. Wang, and M. Flashner, Mechanism of Arthrobacter sialophilus neuraminidase The binding of substrates and transition-state analogs, Biochem. Biophys. Res. Commun., 83 (1978) 1479-1487. 

Kinetic characterization of several selected BsCM variants shows that truncation or mutation of the C-terminal tail has little effect on the turnover number (fcc ll) of the enzyme (Tab. 3.1). When chorismate is bound to the active site of the variants, it is converted to prephenate nearly as efficiently as with wild-type BsCM. However, a substantial reduction in the k /K value is evident (Tab. 3.1). This finding indicates that the C-terminus, while not directly involved in the chemical transformation of bound ligand, does contribute to enzymatic efficiency by uniform binding of substrate and transition state. 

A variety of techniques have been applied to investigate enzyme reaction mechanisms. Kinetic and X-ray crystallographic studies have made major contributions to the elucidation of enzyme mechanisms. Valuable information has been gained from chanical, spectroscopic and biochemical studies of the transition-state structures and intermediates of enzyme catalysis. Computational studies provide necessary refinement toward our understanding of enzyme mechanisms. The ability of an enzyme to accelerate the rate of a chemical reaction derives from the complementarity of the enzyme s active site structure to the activated complex. The transition state by definition has a very short lifetime ( 10 s). Stabilization of the transition state alone is necessary but not sufficient to give catalysis, which requires differential binding of substrate and transition state. Thus a detailed enzyme reaction mechanism can be proposed only when kinetic, chemical and structural components have been studied. The online enzyme catalytic mechanism database is accessible at EzCatDB (http //mbs.cbrc.jp/EzCatDB/). 

Uniform binding of substrate and transition state imparts no extra catalysis... 

The AG for binding the substrate and the transition state is shown as a difference between the energies of the ES complex and E + S. The AG for binding the transition state is shown as a difference between the energies of the E TS complex and E + TS. If the transition state binds tighter (bigger AG) than the substrate, the enzyme-catalyzed reaction must have a lower activation energy. 

As remarked already, kc/ku measures the maximal acceleration at levels of the CD sufficient to saturate complexation of the substrate. By looking carefully at the variations of this ratio with structure one may obtain insights into the mode of transition state binding (VanEtten et al., 1967a,b Bender and Komiyama, 1978). More useful is the ratio k2/ku (=kc/K ku) because it takes into account the effect of substrate binding and it scales the reactivity of S towards the CD to its intrinsic reactivity in the absence of CD. 

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