title: Site-directed mutagenesis of predicted active site residues in glutamate carboxypeptidase II.
abstract: Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate and glutamate. GCP II also hydrolyzes gamma-glutamyl bonds in folylpolyglutamate. The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose crystal structures have been determined. These aminopeptidases are cocatalytic zinc metallopeptidases belonging to the peptidase family M28. Specific zinc and substrate ligands have been proposed in GCP II based on the amino acid sequence alignment to these M28 family members. In the present study, site-directed mutagenesis has been used to test the assignment of these putative ligands in human GCP II. Substitutions to the five putative zinc ligands resulted in severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis. In addition, substitutions of amino acids near the putative zinc ligands have identified other specific residues important for enzyme structure and/or function. Substitutions to putative substrate ligands were less perturbing, and increases in Km were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu). The results from substitutions at the proposed zinc and substrate ligands are consistent with the assignment of these residues and suggest that GCP II has a three-dimensional structure similar to other members of the peptidase family M28.
authors.affiliation: Laboratory of Molecular and Developmental Neuroscience, Massachusetts General Hospital-East, Charlestown, Massachusetts, USA.
authors.forename: H S
authors.initials: HS
authors.lastname: Speno
authors.forename: R
authors.initials: R
authors.lastname: Luthi-Carter
authors.forename: W L
authors.initials: WL
authors.lastname: Macias
authors.forename: S L
authors.initials: SL
authors.lastname: Valentine
authors.forename: A R
authors.initials: AR
authors.lastname: Joshi
authors.forename: J T
authors.initials: JT
authors.lastname: Coyle
mesh_terms: D000954:Antigens, Surface; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000502:physiology; D043425:Glutamate Carboxypeptidase II; D006801:Humans; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D002268:Carboxypeptidases; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D015032:Zinc