Site-Directed Mutagenesis of Predicted Active Site Residues in Glutamate Carboxypeptidase II

• DOI: 10.1124/mol.55.1.179
• Publication Date: 2018-01-08T20:33:08Z
• Authors: Henry S. Speno; Ruth Luthi-Carter; Wendy L. Macias; Stacey L. Valentine; Amit R. T. Joshi; Joseph T. Coyle
• Keywords: Site-directed mutagenesis; Hydrolase; Dipeptidase

Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of neuromodulator<i>N</i>-acetyl-aspartylglutamate to<i>N</i>-acetyl-aspartate and glutamate. GCP also hydrolyzes γ-glutamyl bonds in folylpolyglutamate. The predicted amino acid sequence displays similarities to aminopeptidases from<i>Streptomyces griseus</i> <i>Vibrio proteolyticus</i>, whose crystal structures have been determined. These are cocatalytic zinc metallopeptidases belonging peptidase family M28. Specific substrate ligands proposed based on alignment these M28 members. In present study, site-directed mutagenesis has used test assignment putative human II. Substitutions five resulted severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis. addition, substitutions acids near identified other specific residues important for structure and/or function. were less perturbing, increases <i>K</i>_m observed that introduced a large charge perturbation (e.g., Lys Glu). results from at consistent with suggest three-dimensional similar members