The alpha-chymotryptic ydrolysis of glycine esters.

1. The alpha-chymotrypsin-catalysed hydrolysis of N-acetylglycine ethyl and thiolethyl esters was investigated at pH7.90 and 25 degrees over a wide range of substrate concentrations. 2. 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 10-100mum, with a pseudo-first-order rate constant of approx. 1x10(-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 k(0)/K(m) for the substrate under investigation. 3. 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. 4. Values of K(m) 387mm and k(0) 0.039sec.(-1), and K(m) 41mm and k(0) 0.23sec.(-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. 5. 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.