Reactive cysteine in the active-site motif of Bacteroides thetaiotaomicron dipeptidyl peptidase III is a regulatory residue for enzyme activity.

Dipeptidyl peptidase III (DPP III), a member of the metallopeptidase family M49, was considered as an exclusively eukaryotic enzyme involved in intracellular peptide catabolism and pain modulation. In 2003, new data on genome sequences revealed the first prokaryotic orthologs, which showed low sequence similarity to eukaryotic ones and a cysteine (Cys) residue in the zinc-binding motif HEXXGH. Here we report the cloning and heterologous expression of DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The catalytic efficiency of bacterial DPP III for preferred synthetic substrate hydrolysis was very similar to that of the human host enzyme. Substitution of Cys450 from the active-site motif by serine did not substantially change the enzymatic activity. However, this residue was wholly responsible for the inactivation effect of sulfhydryl reagents. Molecular modeling indicated seven basic amino acid residues in the local environment of Cys450 as a possible cause for its high reactivity. Sequence analysis of 81 bacterial M49 peptidases showed conservation of the HECLGH motif in 68 primary structures with the majority of proteins lacking an active-site Cys originated from aerobic bacteria. Data obtained suggest that Cys450 of B. thetaiotaomicron DPP III is a regulatory residue for the enzyme activity.

Molecular determinants of human dipeptidyl peptidase III sensitivity to thiol modifying reagents.

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.

Human dipeptidyl peptidase III: insights into ligand binding from a combined experimental and computational approach.

Human dipeptidyl peptidase III (DPP III) is a zinc-exopeptidase with implied roles in protein catabolism, pain modulation, and defense against oxidative stress. To understand the mode of ligand binding into its active site, we performed molecular modeling, site-directed mutagenesis, and biochemical analyses. Using the recently determined crystal structure of the human DPP III we built complexes between both, the wild-type (WT) protein and its mutant H568N with the preferred substrate Arg-Arg-2-naphthylamide (RRNA) and a competitive inhibitor Tyr-Phe-hydroxamate (Tyr-Phe-NHOH). The mutation of the conserved His568, structurally equivalent to catalytically important His231 in thermolysin, to Asn, resulted in a 1300-fold decrease of k(cat) for RRNA hydrolysis and in significantly lowered affinity for the inhibitor. Molecular dynamics simulations revealed the key protein-ligand interactions as well as the ligand-induced reorganization of the binding site and its partial closure. Simultaneously, the non-catalytic domain was observed to stretch and the opening at the wide side of the inter-domain cleft became enhanced. The driving force for these changes was the formation of the hydrogen bond between Asp372 and the bound ligand. The structural and dynamical differences, found for the ligand binding to the WT enzyme and the H568N mutant, and the calculated binding free energies, agree well with the measured affinities. On the basis of the obtained results we suggest a possible reaction mechanism. In addition, this work provides a foundation for further site-directed mutagenesis experiments, as well as for modeling the reaction itself.

Absolutely conserved tryptophan in M49 family of peptidases contributes to catalysis and binding of competitive inhibitors.

The role of the unique fully conserved tryptophan in metallopeptidase family M49 (dipeptidyl peptidase III family) was investigated by site-directed mutagenesis on human dipeptidyl peptidase III (DPP III) where Trp300 was subjected to two substitutions (W300F and W300L). The mutant enzymes showed thermal stability equal to the wild-type DPP III. Conservative substitution of the Trp300 with phenylalanine decreased enzyme activity 2-4 fold, but did not significantly change the K(m) values for two dipeptidyl 2-naphthylamide substrates. However, the K(m) for the W300L mutant was elevated 5-fold and the k(cat) value was reduced 16-fold with Arg-Arg-2-naphthylamide. Both substitutions had a negative effect on the binding of two competitive inhibitors designed to interact with S1 and S2 subsites. These results indicate the importance of the aromatic nature of W300 in DPP III ligand binding and catalysis, and contribution of this residue in maintaining the functional integrity of this enzyme's S2 subsite.

Functional tyrosine residue in the active center of human dipeptidyl peptidase III.

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.

Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins.

Dipeptidyl peptidase III (DPP III) is a zinc exopeptidase with an implied role in the mammalian pain-modulatory system owing to its high affinity for enkephalins and localisation in the superficial laminae of the spinal cord dorsal horn. Our study revealed that this human enzyme hydrolyses opioid peptides belonging to three new groups, endomorphins, hemorphins and exorphins. The enzymatic hydrolysis products of endomorphin-1 were separated and quantified by capillary electrophoresis and the kinetic parameters were determined for human DPP III and rat DPP IV. Both peptidases cleave endomorphin-1 at comparable rates, with liberation of the N-terminal Tyr-Pro. This is the first evidence of DPP III acting as an endomorphin-cleaving enzyme.