Characterization of a Reversibly Formed Enzyme Complex in the Reaction of Chymotrypsin with Diisopropyl Fluorophosphate

Abstract

Considerable evidence that the interaction of cr-chymotrypsin with substrates or inhibitors results in conformational changes in the enzyme has been reported (l-6). This evidence was provided in part by experiments with covalently bound enzymesubstrate complexes which indicated that the optical rotatory dispersion parameters (l-3) and the thermodynamic parameters of reversible, temperature-induced phase transitions (4) differ from those of the free enzyme and depend on the nature of the substrate. Other evidence was provided by measurements of difference spectra between enzyme and covalently bound enzymesubstrate complexes (5, 7). The difference spectrum (see, for example, Fig. 3b), with a major peak at 290 rnp, was observed to be the same whether the group attached to the enzyme was diisopropylphosphoryl, acetyl, trimethylacetyl, or cinnamoyl. Recent data on x-ray diffraction from crystals of native y-chymotrypsin and of a covalently bonded inhibitor-enzyme complex also suggest that some deformation of the protein structure accompanies the formation of the enzyme-inhibitor complex (8). In order to understand the interrelationships of the steps which are associated with the spectral changes, the changes in specific rotation of the enzyme, and the catalytic reaction, we have undertaken a kinetic study of the reaction of cY-chymotrypsin with diisopropyl fluorophosphate in the pH range of 2.0 to 10.0 (9). This reaction yields covalcntly bonded diisopropylphosphoryl-oc-chymotrypsin and HF in stoichiometric amounts (10). In this paper we are reporting equilibrium and kinetic studies of this reaction at pH 2.0, including data on the spectral changes, the change in optical rotation, and the phosphorylation and inhibition of the enzyme. We are also reporting on the chymotrypsin-catalyzed hydrolysis at pH 2.0 of a specific substrate, N-acetyl-n-phenylalanine ethyl ester, and on the spectral changes of the enzyme which accompany this reaction.