The interaction of a-chyrnotrypsin with phenylalanine derivatives containing a free a-amino group. Can. J. Biochem. 48,1058Biochem. 48, -1065Biochem. 48, (1970.The action sf or-chymotrypsin on L-and D-phenylalanine ethyl esters (PEE), L-and D-phenylalanine p-nitrobenzyl esters (PNBE), L-phenylalmine methyl and isopropyl esters, and N-methyl-L-phenylalanine methyl ester has been studied using a pH-stat. The D-esters were not hydrolyzed but acted as competitive inhibitors of the hydrolysis of the L-isomers. The N-methyl ester was very slowly hydrolyzed due to its Iow kc,,. For L-PEE (pK 7.23) and L-PNBE (pK 6.931, the activity of a-chymotrypsin is displaced to a more acid region relative to that for the N-acyl amino acid esters. The Km increases sharply below pH 6.5 while the kc,, and k,,,/Km show maxima at pH 6 and 5.6, respectively. On the acid side kc,, is controlled by a basic group of pK 4.86 for L-PNBE and pK 5.1 for L-PEE, and kc,,/Km by a basic group s f p r 6.4 for L-PNBE and pK6.6 for L-PEE. It is proposed that (i) deacylation is rate-limiting, (ii) in the cata1ytlcall.y active entities of the enzyme-substrate complex and acyl enzyme, the a-amino group of the substrate IS protsnated, (iii) the pK of the basic group on the acyl enzyme is considerably lowered by the presence t of the a-NH3 of the substrate, and (iv) the increase in Km and Kf below pH 6.8 is due to the development of unfavorable charge interactions. Introductionan a-acyl amino group by an amino group leads Prior to the work of Petitc1el-c and Benoiton to a partial loss in stereospecificity4 (7)-The (1) on ~-t~r~~i~~ ethyl ester, there had been no present paper describes some steady-state kinetic study of the p H dependence of the parameters studies of the interaction of i*-chymotry~sin k,,,3 and K, for the r*-chymotrypsin-catalyzed with several derivatives of phenylalanine conhydrolysis of free amino acid esters. It had been taining free a-amino groups. reported that the pH optima for the hydrolysis of L-tyrosine ethyl ester, L-tyrosine hydrazide, and L-tyrosine kydroxamide were 6.5, 7.0, and 6.95, respectively (2-41, and kc,, and .Km values had been derived for the latter. Petitclerc (5) has shown that in contrast to the results obtained for N-acyl amino acid esters (ti), the hydrolysis of L-tyrosine ethyl ester is characterized by a strong pH dependence of Km below pH 7 and by a sharp optimum in kc,, at pH 6.1. Moreover, he was unable to detect any hydro~ysis of the D-enantiomer although Hein and Niemann have stated that besides leading to a marked decrease in the rate of hydrolysis, replacement of