The interaction of α-chymotrypsin with phenylalanine derivatives containing a free α-amino group

Abstract

The action of α-chymotrypsin on L- and D-phenylalanine ethyl esters (PEE), L- and D-phenylalanine p-nitrobenzyl esters (PNBE), L-phenylalanine methyl and isopropyl esters, and N-methyl-L-phenylalamne 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 low Kcat. For L-PEE (pK 7.23) and L-PNBE (pK 6.93), the activity of α-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 kcat and kcat/Km show maxima at pH 6 and 7.6, respectively. On the acid side kcat is controlled by a basic group of pK 4.86 for L-PNBE and pK 5.1 for L-PEE, and kcat/Km by a basic group of pK 6.4 for L-PNBE and pK 6.6 for L-PEE. It is proposed that (i) deacylation is rate-limiting, (ii) in the catalytically active entities of the enzyme–substrate complex and acyl enzyme, the α-amino group of the substrate is protonated, (iii) the pK of the basic group on the acyl enzyme is considerably lowered by the presence of the [Formula: see text] of the substrate, and (iv) the increase in Km and Ki below pH 6.8 is due to the development of unfavorable charge interactions.