THE ACTIVITY OF CARBOXYPEPTIDASE-Y TOWARD SUBSTRATES WITH BASIC P-1 AMINO-ACID-RESIDUES IS DRASTICALLY INCREASED BY MUTATIONAL REPLACEMENT OF LEUCINE-178

A random mutagenesis study on carboxypeptidase Y has previously sugges ted that Leu178 is situated in the S1 binding pocket, and this has lat er been confirmed by the three-dimensional structure. We here report t he mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using su bstrates systematically varied at the P-1 position. The general effect of these substitutions is a reduced k(cat)/K-m for substrates with un charged amino acid residues in the P-1 position, little effect on thos e with acidic residues, and an increased k(cat)/K-m for those with bas ic amino acid residues. There is a clear correlation between the reduc tion in k(cat)/K-m for substrates with uncharged P-1 side chains and t he nature of the residue at position 178. A small reduction is observe d when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very l arge reduction when it is replaced by a charged residue. When Leu178 i s replaced by Asp, k(cat)/K-m is reduced by a factor of 2200 for a sub strate with Val in the P-1 position. The k(cat)/K-m values for the hyd rolysis of substrates with charged P-1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge. Surprisingly, all mutants (except L178K) exhibit increased act ivity with substrates with basic P-1 side chains. The largest increase in k(cat)/K-m for substrates with basic P-1 side chains is observed w ith polar or acidic amino acid residues at position 178; e.g., with Se r and Asp k(cat)/K-m for a substrate with Lys in the P-1 position is i ncreased relative to the wild type by factors of 70 and 90, respective ly. On the basis of all the kinetic data it is hypothesized that this increase in activity is obtained by elimination of unfavorable steric constraints present in the wild-type enzyme rather than by introductio n of favorable binding contributions in the mutants. The enzyme that h as Leu178 replaced by Asp exhibits the largest increase (Lys in P-1) a s well as the largest decrease (Val in P-1) in k(cat)/K-m values, and as a consequence the relative k(cat)/K-m value has been changed by a f actor of 2 x 10(5). It is demonstrated that the L178S and L178D enzyme s, due to their altered substrate preference, can be applied in the de termination of C-terminal sequences of peptides containing basic amino acids in the C-terminal region.