The α-chymotryptic hydrolysis of glycine esters

Abstract

The [alpha]-chymotrypsin-catalyzed hydrolysis of N-acetylglycine ethyl and thiol-ethyl esters was investigated at pH 7-90 and 25[degree] over a wide range of substrate concentrations. The Lineweaver-Burk plots for these substrates are markedly curved, and it is shown that the curvature is due solely to the "enzyme-blank" reaction. The rate of this reaction is proportional to free enzyme concentration in the range 10-100 [mu][image], with a pseudo-1st-order rate constant of approx. 1 x 10-3 sec.-1. Correction for this reaction by the procedure described leads to linear plots. It is shown that the significance of the enzyme-blank reaction depends on the value of ko/Km for the substrate under investigation. Interpretation of the curvature in the Lineweaver-Burk plots by previous workers in terms of activation by excess of substrate is shown to be erroneous. Values of Km 387 m[image] and ko 0-039 sec.-1, and Km 41 m[image] and ko 0.23 sec.-1, were obtained for the ethyl and thiolethyl esters of N-acetylglycine respectively. The literature values for the methyl esters of N-acetyl- and N-propionyl-glycine have been corrected by the procedure described. The new values agree much better with current theories of [alpha]-chymotrypsin mechanism and specificity. The kinetic parameters for the ethyl and thiolethyl esters indicate the absence of an electrophilic component in the catalytic mechanism of [alpha]-chymotrypsin, and the importance of the ester function in substrate binding.