SITE-DIRECTED MUTAGENESIS ON (SERINE) CARBOXYPEPTIDASE-Y FROM YEAST -THE SIGNIFICANCE OF THR60 AND MET398 IN HYDROLYSIS AND AMINOLYSIS REACTIONS

In (serine) carboxypeptidase Y, the flexible side chain of Met398 form s one side of the S-1' binding pocket and the beta- and gamma-carbon a toms of Thr60 form the opposite side. Met398 has been substituted with the residues Gly, Ala, Val, Ile, Leu, Phe, and Tyr while Thr60 has be en substituted with the residues Ala, Val, Leu, Met, Phe, and Tyr by s ite-directed mutagenesis, and the resulting enzymes have been characte rized with respect to their P-1' substrate preferences using the subst rate series FA-Phe-Xaa-OH (Xaa = Gly, Ala, Val, or Leu) and FA-Ala-Yaa -OH (Yaa = Leu, Gin, Glu, Lys, or Arg). The results show that Met398 i s much more important for transition state stabilization than Thr60 al though it appears that the selected nonbulky amino acid residue (Thr) at position 60 is important for high k(cat) values. The results furthe r suggest that bulky amino acid side chains at position 398 are able t o adjust the size of the S-1' pocket such that favorable interactions with the substrate can be obtained with even small P-1' side chains, e .g., Gly. Accordingly, the hydrolysis of substrates with bulky/hydroph obic P-1' side chains is less dependent on the nature of the amino aci d residue at position 398 than that of a substrate with a nonbulky P-1 ' side chain. The three-dimensional structure of the mutant enzyme E65 A + E145A has been determined, and it provides support for the high mo bility of the Met398 side chain. In transpeptidation reactions the sub stitutions at position 398 also influence the interactions between the binding pocket and the amino acid leaving group as well as the added nucleophile competing with water in the deacylation reaction. Much hig her aminolysis was obtained with some of the mutant enzymes, presumabl y due to a changed accessibility of water to the acyl-enzyme intermedi ate while the nucleophile/leaving group is bound at the S-1' binding s ite.