Naslov
Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III

Autori
Abramić, Marija ; Šimaga, Šumski ; Osmak, Maja ; Čičin-Šain, Lipa ; Vukelić, Bojana ; Vlahoviček, Kristian ; Dolovčak, Ljerka

Izvornik
The International Journal of Biochemistry & Cell Biology (1357-2725) 36 (2004); 434-446

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
dipeptidyl peptidase III; reactive SH-groups; oxidant; redox regulation; rat tissues
(dipeptidyl pepitdase III; reactive SH-groups; oxidant; redox regulation; rat tissues)

Sažetak
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 +/- 567 M-1min-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 3540 M-1sec-1 for slower reacting sulfhydryl, and 21855 M-1sec-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 H2O2 inactivated the rat DPP III. Human enzyme was resistant to H2O2 attack and less affected by reduced glutathione 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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija, Temeljne medicinske znanosti

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