engleski

MOLECULAR BASIS OF HUMAN DIPEPTIDYL PEPTIDASE III SUSCEPTIBILITY TO SULFHYDRYL REAGENTS

1. Introduction: Human dipeptidyl peptidase III (DPP III) is an M49 family metallopeptidase. All characterized members of this family are inhibited by thiol modifying reagents and all contain multiple cysteins. Human DPP III has six cysteines, three in the upper and three in the lower domain. Since there are no invariant cysteines within the M49 family, the identity of the residues responsible for the enzyme inactivation seems to be species specific. Here we present the results on the determination of molecular basis of human DPP III sensitivity to thiol modifying reagents using mutational analysis and mass spectrometry (MS)[1]. 2. Results and Discussion: We treated the wild-type human DPP III and six single Cys-to-Ala mutants with five SH group modifying reagents and measured the residual activity. Cys176 was identified as the residue most responsible for DPP III inactivation with organomercurial p-hydroxymercuribenzoate (pHMB). Other results indicated differing reactivity of cysteine residues with diverse thiol-modifying compounds. Cysteine residues at positions 19, 176, 509 and 654 contributed to enzyme inactivation with aromatic disulfides. Also, the results obtained upon treatment with GSSG showed that inactivation with this reagent is due to modification of more than one cysteine residue. MS analysis of glutathionylated wild-type showed partial glutathionylation of three residues: Cys176, Cys654 and Cys147, but the residual activity assay confirmed that Cys176 and Cys654 have a role in inactivation by GSSG. Our results indicate that human DPP III cysteine residues may participate in biological reactions employing reactive sulfhydryls, and may be important for functions other than proteolysis. The finding that Cys176 (a residue from the lower domain, whereas the active site is in the upper domain) is involved in inactivation with all investigated reagents shows that both domains form the substrate binding site. [1] Karačić, Z., Špoljarić, J., Rožman, M., and Abramić, M. (2012) Molecular determinants of human dipeptidyl peptidase III sensitivity to thiol modifying reagents. Biol. Chem. 393 , 1523 – 1532.

dipeptidyl peptidase III; glutathionylation; mass spectrometry; reactive cysteine; sulfhydryl reagent

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