Application of quantitative structure-activity relationship modeling to the evaluation of the changes in enzymatic activity of carboxypeptidase Y upon chemical modifications.

A series of 18 phenacyl bromide and iodoacetamide analogues have been synthesized and used to alkylate Met-398 situated in the S'1 binding site of carboxypeptidase Y. The course of the reactions was monitored by measurements of the peptidase and esterase activities. All except four of the reagents reacted selectively, and from these preparations the modified enzymes were purified and kinetically characterized toward a methyl ester substrate and a peptide substrate with a large leaving group in the P'1 position. The Km, kcat, and kcat/Km for the hydrolysis of these substrates have been quantitatively correlated to parameters describing the properties of the modification reagents. The esterase activity depends only on the steric bulk of the para-substituents with the phenacyl-modified enzymes, but on both steric and electronic factors of the N-alkyl substituents with the acetamide modified enzymes. The peptidase activity, on the other hand, is dependent on steric and electronic factors with both types of modified enzymes.