EFFECTS OF INTRODUCED ASPARTIC AND GLUTAMIC-ACID RESIDUES ON THE P'(1)) SUBSTRATE-SPECIFICITY, PH-DEPENDENCE AND STABILITY OF CARBOXYPEPTIDASE-Y

Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharo myces cerevisiae with a preference for C-terminal hydrophobic amino ac id residues. In order to alter the inherent substrate specificity of C PD-Y into one for basic amino acid residues in P-1', we have introduce d Asp and/or Glu residues at a number of selected positions within the Si binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain s ignificant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite diff erent specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (k(cat)/K-m)(FA-A-R-OH)/(k(cat)/K-m)(FA-A -L-OH) ratio. The change in stability upon introduction of Asp/Glu res idues can be correlated to the difference in the mean buried surface a rea between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent acc essible surface as defined by the surrounding residues in the wild typ e enzyme or is submerged below.