Site-directed mutagenesis of predicted active site residues in glutamate carboxypeptidase II

Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolys is of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate a nd glutamate. GCP II also hydrolyzes gamma-glutamyl bonds in folylpolygluta mate. The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose c rystal structures have been determined. These aminopeptidases are cocatalyt ic zinc metallopeptidases belonging to the peptidase family M28. Specific z inc and substrate ligands have been proposed in GCP II based on the amino a cid sequence alignment to these M28 family members. In the present study, s ite-directed mutagenesis has been used to test the assignment of these puta tive ligands in human GCP II. Substitutions to the five putative zinc ligan ds resulted in severely reduced enzyme activity, although mutant protein wa s expressed as demonstrated by immunoblot analysis. In addition, substituti ons of amino acids near the putative zinc ligands have identified other spe cific residues important for enzyme structure and/or function. Substitution s to putative substrate ligands were less perturbing, and increases in K-m were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu). The results from substitutions at the proposed zinc an d substrate ligands are consistent with the assignment of these residues an d suggest that GCP II has a three-dimensional structure similar to other me mbers of the peptidase family M28.