Site-directed mutagenesis on (serine) carboxypeptidase Y. A hydrogen bond network stabilizes the transition state by interaction with the C-terminal carboxylate group of the substrate

The three-dimensional structure of (serine) carboxypeptidase Y suggests that the side chains of Trp49, Asn51, Glu65, and Glu145 could be involved in the recognition of the C-terminal carboxylate group of peptide substrates. The mutations Trp49 to Phe; Asn51 to Ala, Asp, Glu, Gln, Ser, or Thr; Glu65 to Ala; and Glu145 to Ala, Asp, Asn, Gln, or Ser have been performed. Enzymes with Ala at these positions were also produced as double and triple mutations. These mutations have only little effect on the esterase activity of the enzyme, consistent with the absence of a hydrogen bond acceptor in the P1' position of such substrates. On the other hand, removal of the hydrogen-bonding capacity by incorporation of Ala at any of these four positions results in reduced peptidase activity, in particular when Asn51 and Glu145 are replaced. The results are consistent with Trp49 and Glu65 orienting Asn51 and Glu145 by hydrogen bonds, such that these can function as hydrogen bond donors (Glu145 only in its protonated carboxylic acid form) with the C-terminal alpha-carboxylate group of the peptide substrate as acceptor. However, it appears that strong interactions are formed only in the transition state since the combined removal of Asn51 and Glu145 reduces kcat about 100-fold and leaves K(M) practically unchanged. The results obtained with enzymes in which Asn51 or Glu145 has been replaced with other residues possessing the capacity to donate a hydrogen bond demonstrate that there is no flexibility with respect to the nature of the hydrogen bond donor at position 145, whereas enzymes with Gln, Ser, or Thr at position 51 exhibit much higher activity than N51A, although none of them reaches the wild-type level. With carboxypeptidase Y as well as other serine carboxypeptidases the binding of peptide substrates in the ground state (K(M)) is adversely affected by an increase in pH