On the Specificity of Elastase. Hydrolysis of Peptide p-Nitrobenzyl Esterst

Five series of p-nitrobenzyl esters of peptides having the general formula Alan-1X-ONbz (where n = 2, 3, and 4 and X = Ala, Gly, Val, Phe, or Leu) were synthesized and subjected to enzymic hydrolysis by porcine elastase. All substrates were split at the ester bond. Km and kcat values were determined for all but two of the substrates. kcat/jKm, values increased strongly with n in all series, indicating a specificity with respect to the X residue: Ala > Leu > Val > Gly > Phe, except for n = 4, where Ala Leu. Judged from Km values, strength of binding is strongly size dependent and follows Ala > Val > Leu (when n > 2). kmt was hardly dependent on peptide length for X = Ala (60-100 sec-1) and X = Val (10-20 sec-1) but for X = Leu it increased dramatically from 20 sec-1 (n = 2) to 213 sec-1 (n = 3) and 625 sec-1 (n = 4). It is suggested that this phenomenon is caused by strain in the “Michaelis complex”-due to the strong binding of the ONbz group-facilitating acylation of the enzyme. Support for this view is found in the observation that in the hydrolysis of peptide bonds in the pair Ala3Ala-LysAla/Ala3Leu-LysAla (at the bond), Ala is favored 50-fold over Leu.