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Molecular determinants of human dipeptidyl peptidase III sensitivity to thiol modifying reagents
Zrinka Karačić1, Jasminka Špoljarić, Marko Rožman
1Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, Zagreb, Croatia.
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.
Insights
Human dipeptidyl peptidase III (DPP III) susceptibility to sulfhydryl reagents was investigated. Cysteine 176, distant from the active site, is key to DPP III inactivation, suggesting a broader substrate-binding site.
Area of Science:
- Biochemistry
- Enzymology
- Metallopeptidases
Background:
- Human dipeptidyl peptidase III (DPP III) is a metallopeptidase involved in protein metabolism and oxidative stress.
- DPP III has a bilobal structure with six cysteine residues crucial for its function.
- Understanding cysteine reactivity is vital for elucidating enzyme mechanisms and drug interactions.
Purpose of the Study:
- To investigate the molecular basis of human DPP III susceptibility to sulfhydryl reagents.
- To identify specific cysteine residues involved in enzyme inactivation by various reagents.
- To explore the role of distant cysteines in enzyme activity and substrate binding.
Main Methods:
- Biochemical analysis of cysteine-to-alanine mutants of human DPP III.
- Mass spectrometry to identify modified cysteine residues.
- Enzyme activity assays using sulfhydryl reagents and oxidized glutathione (GSSG).
Main Results:
- Cysteine 176 (Cys176) was identified as the primary site of inactivation by organomercurial compounds.
- Cys176 and Cys654 were confirmed to be involved in GSSG-induced inactivation.
- Glutathionylation occurred at Cys147, Cys176, and Cys654 upon GSSG treatment.
Conclusions:
- Human DPP III's susceptibility to sulfhydryl reagents is mediated by specific cysteine residues, including Cys176.
- The involvement of Cys176 in inactivation suggests the substrate-binding site extends beyond the catalytic domain.
- These findings provide insights into the structural and functional properties of human DPP III and its interactions with reactive species.
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