Ultrasonic studies of proton‐transfer reactions at the catalytic site of α‐chymotrypsin

Abstract

Ultrasonic relaxation measurements for ~-chymotrypsin in phosphate, sulfite and arsenate buffers exhibit a high peak of absorption at neutral pH. The analysis is based on: (i) comparison of the relaxation measurements for the enzyme and for the zymogen and inhibited enzyme; (ii) X-ray and neutron diffraction data, and high-resolution NMR data. The ultrasonic relaxation is shown to result mainly from a proton-transfer reaction that involves the histidine at the catalytic site (His-57). The question is raised of whether the enhanced ultrasonic effect observed in the enzyme is indicative of a property that plays a part in the catalytic activity.

Ultrasonic Cole-Cole diagram for solutions and application to alpha-chymotrypsin

“…In the experiments with a-chymotrypsin the ultrasonic absorption was larger in the enzyme than in the zymogen and in the inhibited enzyme, by factors of 8 and 3 respectively. These results led to the conclusion that a proton-transfer reaction involving the histidine at the catalytic site is the primary source for the ultrasonic relaxation in a-chymotrypsin at neutral pH (5).…”

“…The simultaneous exploitation of ultrasonic absorption and velocity data described here was stimulated by recent work with biological molecules and supramolecular assemblies. Among the results of interest are: (a) the observation in viruses and viral capsids of relaxation effects that are specific to the assembled system (1)(2)(3)(4); (b) the observation in a-chymotrypsin of an ultrasonic absorption that is large at neutral pH (5), contrary to most known absorption effects in proteins, whose peak values occur at acidic or basic pH.…”

Ultrasonic Monitoring of Biocatalysis in Solutions and Complex Dispersions

“…Ionizable groups of amino acid side chains with pK a values close to pH 7.8, which are exposed to contact with water as a result of hydrolysis, can also participate in the proton exchange with the ions of the phosphate buffer [85][86][87]. For common experimental conditions the relaxation frequency of the process of proton transfer is expected to be within low MHz range [85][86][87][88][89]. This agrees with our results presented in Figure 7C according to which at frequencies below 10 MHz (approximately) the amplitude of velocity rise during the process depends on the frequency, and a substantial increase of α f 2 during the reaction is observed.…”