title: Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities.
abstract: Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave β-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body.
  
  
  
  The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Gilead Sciences and IOCB Research Centre, Academy of Sciences of the Czech Republic, Prague, Czech Republic.|Department of Biochemistry, Faculty of Natural Sciences, Charles University in Prague, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Navrátil
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Gilead Sciences and IOCB Research Centre, Academy of Sciences of the Czech Republic, Prague, Czech Republic.|Department of Biochemistry, Faculty of Natural Sciences, Charles University in Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Tykvart
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Gilead Sciences and IOCB Research Centre, Academy of Sciences of the Czech Republic, Prague, Czech Republic.|Department of Biochemistry, Faculty of Natural Sciences, Charles University in Prague, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Schimer
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Gilead Sciences and IOCB Research Centre, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Pachl
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Gilead Sciences and IOCB Research Centre, Academy of Sciences of the Czech Republic, Prague, Czech Republic.|Department of Biochemistry, Faculty of Natural Sciences, Charles University in Prague, Czech Republic.
authors.forename: Václav
authors.initials: V
authors.lastname: Navrátil
authors.affiliation: Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Tibor András
authors.initials: TA
authors.lastname: Rokob
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Gilead Sciences and IOCB Research Centre, Academy of Sciences of the Czech Republic, Prague, Czech Republic.|Department of Biochemistry, Faculty of Natural Sciences, Charles University in Prague, Czech Republic.
authors.forename: Klára
authors.initials: K
authors.lastname: Hlouchová
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Gilead Sciences and IOCB Research Centre, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Lubomír
authors.initials: L
authors.lastname: Rulíšek
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Gilead Sciences and IOCB Research Centre, Academy of Sciences of the Czech Republic, Prague, Czech Republic.|Department of Biochemistry, Faculty of Natural Sciences, Charles University in Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Konvalinka
mesh_terms: D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D043425:Glutamate Carboxypeptidase II / Q000737:chemistry / Q000378:metabolism*; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D000954:Antigens, Surface; D005971:Glutamates; C018491:beta-citrylglutamic acid; D002268:Carboxypeptidases; C543915:NAALAD2 protein, human; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II
keywords: GCPIII; QM/MM calculations; arene-binding site; prostate-specific membrane antigen; β-citryl-l-glutamate