INCREASE OF THE P-1 LYS LEU SUBSTRATE PREFERENCE OF CARBOXYPEPTIDASE-Y BY RATIONAL DESIGN BASED ON KNOWN PRIMARY AND TERTIARY STRUCTURES OFSERINE CARBOXYPEPTIDASES/

The P-1 substrate preference of serine carboxypeptidases, as expressed by the Lys/Leu ratio, differs by up to 10(5)-fold, Predictions of the major determinants of this preference are made by correlating primary and tertiary structures to substrate preferences, In carboxypeptidase Y from yeast it is predicted that Trp312 constitutes such a determina nt, reducing the P-1 Lys/Leu substrate preference of this enzyme. The predictions are tested by the construction and kinetic characterizatio n of ten mutant enzymes of carboxypeptidase Y. All of these enzymes ex hibit changes in their Pi substrate preference. Generally, small decre ases in activity (k(cat)/K-m) are observed with substrates containing uncharged P-1 side chains. With substrates containing acidic P-1 side chains, i.e., FA-Glu-Ala-OH, the activity generally increases slightly , 7-fold in the case of W312K. The most dramatic effects of the Trp312 substitutions are observed with substrates containing basic P-1 side chains, i.e., k(cat)/K-m for the hydrolysis of FA-Lys-Ala-OH with W312 E has increased 1150-fold, exclusively as a result of increased k(cat) values. Similar results have previously been obtained by mutational s ubstitution at position 178 of carboxypeptidase Y. The construction an d kinetic characterization of position 178+312 double mutants demonstr ate that the kinetic effects of substitutions at these two positions a re not additive. The P-1 Lys/Leu substrate preference of one double mu tant, L178D+W312D, has changed 380000-fold as compared to the wild typ e enzyme, and the overall P-1 substrate preference of this enzyme clos ely resembles that of carboxypeptidase WII from wheat.