Comparative studies of the specificities of α-chymotrypsin and subtilisin BPN′. Studies with flexible substrates

A series of arylalkanoate esters and alpha-acetamidoarylalkanoate esters were tested as substrates for alpha-chymotrypsin and subtilisin BPN'. Chymotrypsin hydrolysed N-acetyl-l-phenylalanine methyl ester and methyl 4-phenylbutyrate faster than their respective higher and lower homologues, whereas methyl 2-acetamido-6-phenylhexanoate and methyl 6-phenylhexanoate were better substrates for subtilisin than their lower homologues. N-Acetyl-l-tryptophan methyl ester and its analogue, N-acetyl-3-(1-naphthyl)-alanine methyl ester, were hydrolysed 23 times faster by chymotrypsin than by subtilisin. These results indicate that the binding site of alpha-chymotrypsin is roughly 1.1nm (11A) long and curved, whereas that of subtilisin is a longer system and less curved. The stereo-specificity during the hydrolysis of typical substrates by both enzymes was found to vary over a wide range. The enhancing effect of the alpha-acetamido group in the l-series of substrates and the detrimental effect in the d-series of substrates also varies considerably.