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 forms one side of the S sub(1)' binding pocket and the beta - and gamma -carbon atoms of Thr60 form the opposite side. Met398 has been substituted with the residues Gly, Ala, Val, Ile, Leu, Phe, and Tyr while Thr60 has been substituted with the residues Ala, Val, Leu, Met, Phe, and Tyr by site-directed mutagenesis, and the resulting enzymes have been characterized with respect to their P sub(1)' substrate preferences using the substrate series FA-Phe-Xaa-OH (Xaa = Gly, Ala, Val, or Leu) and FA-Ala-Yaa-OH (Yaa = Leu, Gln, Glu, Lys, or Arg). The results show that Met398 is much more important for transition state stabilization than Thr60 although it appears that the selected nonbulky amino acid residue (Thr) at position 60 is important for high k sub(cat) values. The results further suggest that bulky amino acid side chains at position 398 are able to adjust the size of the S sub(1)' pocket such that favorable interactions with the substrate can be obtained with even small P sub(1)' side chains, e.g., Gly. Accordingly, the hydrolysis of substrates with bulky/hydrophobic P sub(1)' side chains is less dependent on the nature of the amino acid residue at position 398 than that of a substrate with a nonbulky P sub(1)' side chain. The three-dimensional structure of the mutant enzyme E65A + E145A has been determined, and it provides support for the high mobility of the Met398 side chain. In transpeptidation reactions the substitutions 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 higher aminolysis was obtained with some of the mutant enzymes, presumably due to a changed accessibility of water to the acyl-enzyme intermediate while the nucleophile/leaving group is bound at the S sub(1)' binding site.