The specificity in the elementary steps of α-chymotrypsin catalysis A temperature study with a series of N-acetyl-l-amino acid methyl esters

Abstract The method of ‘added nucleophile’ (methanol) was employed to study the steady-state kinetics of the α-chymotryptic hydrolysis of methyl esters of N- acetyl- l -amino acids of the RCH(NHCOCH 3 )-C(O)OCH 3 type, which are derivatives of α-aminobutyric acid, norvaline, norleucine, phenylalanine, α-aminoheptanoic acid and α-aminooctanoic acid. In the 5 to 30°C temperature range at pH 8.0 the elementary rate and binding constants for both hydrolysis ( K s , k 2 , k 3 ) and methanolysis ( k –2 ) of the intermediate acylenzyme have been determined. The following results are discussed: 1. The enthalpy-entropy compensation phenomenon, which has previously been established to take place in α-chymotryptic catalysis, is characterized by two values of isokinetic temperatures ( T c ), i.e., about 200 K at the step of binding the substrate and 430–460 K at the chemical steps of the enzymafic reaction. 2. It is demonstrated how the free energy and enthalpy, as well as entropy, change along all the reaction coordinate. To explain the results obtained, a mechanism for enzyme-substrate interaction is suggested according to which the sorption of substrate group R on the enzyme is different in the metastable intermediates and in the transition states of the reaction: (i) in the enzyme-substrate complex and in the acylenzyme, substrate group R merely emerges from water to be buried in the protein. Two facts point to the hydrophobic nature of the E-R interacfion. First, the change in the free energy is exactly ΔG extr R , that is the free energy of the transfer (extraction) of substrate group R from water to the organic solvent. Second, the motive force of the E-R interaction is the change in the entropy only. (ii) In the transition state the situation is different, i.e., the E-R interaction becomes thermodynamically still more favourable (the free energy change is 2 ΔG trans R ) and involves thermodynamically favourable changes not only in the entropy (as is the case in the intermediates), but also in the enthalpy. Hence the E-R interaction in the transition states is accompanied by conformational and solvational changes of the enzyme-substrate system and, first and foremost, of the entire protein.