title: Molecular determinants of human dipeptidyl peptidase III sensitivity to thiol modifying reagents.
abstract: Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49, involved in protein metabolism and oxidative stress response. DPPIII crystal structure shows the two lobe-like domains separated by a wide cleft. The human enzyme has a total of six cysteines, three in the lower (Cys19, Cys147,and Cys176) and three in the upper (Cys509, Cys519,and Cys654), catalytic, domain containing the active site zinc ion. To elucidate the molecular basis of this enzyme ’ s susceptibility to sulfhydryl reagents, biochemical analysis of a set of Cys to Ala mutants was used, supported by mass spectrometry. Cys176, a residue 44 A apart from the catalytic center of the ligand-free enzyme, was found responsible for the inactivation with the submicromolar concentration of an organomercurial compound, and three additional cysteines contributed to sensitivity to aromatic disulfides. Upon treatment with oxidized glutathione [glutathione disulfide(GSSG)], cysteine residues at positions 147, 176, and 654 were found glutathionylated. The mutational analysis confirmed the involvement of Cys176 and Cys654 inhuman DPP III inactivation by GSSG. Observation that Cys176, a residue quite distant from the active center,contributes to enzyme inactivation, indicates that the substrate-binding site of human DPP III comprises both lower and upper protein domain.
authors.affiliation: Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, Zagreb, Croatia.
authors.forename: Zrinka
authors.initials: Z
authors.lastname: Karačić
authors.forename: Jasminka
authors.initials: J
authors.lastname: Špoljarić
authors.forename: Marko
authors.initials: M
authors.lastname: Rožman
authors.forename: Marija
authors.initials: M
authors.lastname: Abramić
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013439:Sulfhydryl Reagents / Q000378:metabolism*
chemical_list: D013439:Sulfhydryl Reagents; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D003545:Cysteine; D019803:Glutathione Disulfide