Sorensen_1995_J.Am.Chem.Soc_117_5944

In (serine)carboxypeptidase Y, the flexible side chain of Met 398 forms one side of the Si' 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,lie,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 Pi' substrate preferences using thes ubstrate series FA-Phe-Xaa-OH (Xaa=Gly,Ala,Val,orLeu) and FA-Ala-Yaa-OH (Yaa=Leu,Gin,Glu,Lys,or Arg). The results show that Met398 is much more important for transition state stabilization than Thr60 although itappears that the selected non bulky amino acid residue(Thr) at position 60 is important for high Kcat values. The results further suggest that bulky amino acid side chains at position 398 are able to adjust the size of the Si' pocket such that favorable interactions with the substrate can be obtained with even small Pi' side chains,e.g., Gly. Accordingly,the hydrolysis of substrates with bulky/hydrophobic Pi' side chains is less dependent on the nature of the amino acid residue at position 398 than that of a substrate with a non bulky Pi' sid echain.The three-dimensional structure oft hemutant enzymeE65A+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 isbound at the Si' binding site.