title: Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis.
abstract: Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1' site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N-acetyl-l-aspartyl-l-glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1' pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the 'fine-tuning' of GCPII substrate specificity.
authors: Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors: Petra
authors: P
authors: Mlcochová
authors: Anna
authors: A
authors: Plechanovová
authors: Cyril
authors: C
authors: Barinka
authors: Daruka
authors: D
authors: Mahadevan
authors: Jose W
authors: JW
authors: Saldanha
authors: Lubomír
authors: L
authors: Rulísek
authors: Jan
authors: J
authors: Konvalinka
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D043425:Glutamate Carboxypeptidase II / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D051381:Rats; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000954:Antigens, Surface; D018698:Glutamic Acid; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II