Conformational Change Associated with the Acylation of α-Chymotrypsin by N-acetyl-l-phenylalanine Methyl Ester

Abstract

Abstract The reaction of α-chymotrypsin with N-acetyl-l-phenylalanine methyl ester has been studied using subzero temperatures and 65% aqueous dimethylsulfoxide as solvent. Since this substrate is essentially transparent in the region of tryptophan absorption (290 nm), the reaction was monitored by measuring the fluorescence emission of the enzyme. By initiating the reaction at -80 or -90° (in the pH range 4 to 7.5), three sequential reactions could be detected as the temperature was progressively raised. The first reaction was attributed to binding of substrate, the latter two to acylation and deacylation, respectively. Comparison of the fluorescence emission spectra of the products of these reactions with enzyme alone under identical conditions was made. The spectrum of the product of the first reaction differed only slightly from free enzyme, whereas the spectrum of the acyl-enzyme had a very substantially reduced emission intensity as well as a blue shift in λmax. The data suggest significant changes in the environment about 1 or more tryptophan residues in the acyl-enzyme as compared to the free enzyme or Michaelis complex.