On the size of the active site in proteases II. Carboxypeptidase-A

Abstract

The size of the active site of carboxypeptidase-A was investigated by studying the kinetics of hydrolysis of peptides of L-alanine, D-alanine and L-phenylalanine, as well as of a number of their N-benzyloxycarbonyl, N-acetyl, N-phenylproprionyl and N-methyloxycarbonyl derivatives. From a comparison of the various kinetic parameters ( K m, k cat) it was concluded that the active site of this enzyme extends over about 18 Å. The binding area can be divided into 5 “subsites”, each accommodating one amino acid residue (or blocking group) of the substrate. By comparing K m values of pairs of substrates containing either a methyl or a benzyl side-chain in equivalent positions, it was shown that the binding area as a whole has a larger affinity towards the aromatic residues. Substitution of a D-residue for an L-residue reduced k cat values rather than K m. In addition a remarkable affinity for the urethane-grouping located specifically at subsite S 3 was found. A methyloxycarbonyl or benzyloxy — carbonyl group occupying this subsite caused a 5-fold increase in K m as compared with an acetyl or phenylproprionyl group, respectively. Methyloxycarbonyl or benzyloxycarbonyl groups in subsites S 2 or S 4 showed no such effect.