title: Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III.
abstract: Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance. The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of the enzyme's residual activity with fluorimetric detection. Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3523+/-567M(-1)min(-1). Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540M(-1)s(-1) for slower reacting sulfhydryl, and 21,855M(-1)s(-1) for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H(2)O(2) inactivated the rat DPP III. Human enzyme was resistant to H(2)O(2) attack and less affected by reduced glutathione (GSH) than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues. These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III.
authors.affiliation: Department of Organic Chemistry and Biochemistry, Ruder Bosković Institute, Bijenicka 54 pp 180, 10002 Zagreb, Croatia. abramic@rudjer.irb.hr
authors.forename: Marija
authors.initials: M
authors.lastname: Abramić
authors.forename: Sumski
authors.initials: S
authors.lastname: Simaga
authors.forename: Maja
authors.initials: M
authors.lastname: Osmak
authors.forename: Lipa
authors.initials: L
authors.lastname: Cicin-Sain
authors.forename: Bojana
authors.initials: B
authors.lastname: Vukelić
authors.forename: Kristian
authors.initials: K
authors.lastname: Vlahovicek
authors.forename: Ljerka
authors.initials: L
authors.lastname: Dolovcak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D005978:Glutathione; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D016415:Sequence Alignment; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D004151:Dipeptides; D006902:Hydroxymercuribenzoates; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C030601:4-hydroxymercuribenzoate; C040852:arginylarginine 2-naphthylamide; D006861:Hydrogen Peroxide; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D005978:Glutathione; D003545:Cysteine