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, G1u65, and G1u145 could be involved in the recognition of the C-terminal carboxylate group of peptide substrates. The mutations Trp49→Phe; Asn51→Ala, Asp, Glu, Gln, Ser, or Thr; Glu65→Ala; and Glu145→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 P 1 ' position of such substrates