Marija Abramić

Substrate specificity and effects of water-miscible solvents on the activity and stability of extracellular lipase from Streptomyces rimosus

Abstract Substrate specificity, regioselectivity and transesterification activity of purified extracellular lipase from Streptomyces rimosus were investigated. The enzyme showed pronounced lipolytic activity toward a number of triacylglycerols and oils of vegetable and animal origin. It hydrolyzed most efficiently medium chain length fatty acid glycerol esters (C8–C12). There was preference for the esters of C16 and C18 unsaturated fatty acids over C16 and C18 saturated fatty acid esters, as well as for triacylglycerol substrate with cis double bond (triolein) versus trans double bond (trielaidin). Streptomyces rimosus lipase hydrolyzed primary and secondary ester bonds in triacylglycerols (triolein and 2,3-dimercapto-1-propanol tributyrate). The lipase catalyzed the hydrolysis of poly(oxyethylene) sorbitan monoesters (Tween 20–80) with rate comparable for that determined with triacylglycerols and oils. Several water-miscible solvents enhanced the lipase activity. 1,4-Dioxane activated the enzyme in a broad concentration range, up to 4-fold. Lipase was stable in solvent mixtures containing 50% (v/v) ethanol, 1,4-dioxane, acetonitrile or acetone. Tetrahydrofuran and N,N -dimethylformamide (both 50%) inactivated the enzyme with t 1/2 of 5 min and t 1/2 of 2 h, respectively. Transesterification of racemic 1-phenyl ethanol with vinyl acetate, catalyzed by extracellular lipase from Streptomyces rimosus in n -hexane, proceeded with partial R -enantioselectivity.

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

Abstract 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.

Human and rat dipeptidyl peptidase III: biochemical and mass spectrometric arguments for similarities and differences.

Abstract Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82 500 ± 60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co2+, differing in their pH optima and kcat /Km . Zn2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III (Ki <1μM) and a preference for a hydrophobic residue at the P1 site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.

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

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 enzymes 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.

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 k cat 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.

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 enzymes S2 subsite.

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

Abstract 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.

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 kcat 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. Copyright

Novel dipeptidyl hydroxamic acids that inhibit human and bacterial dipeptidyl peptidase III

Abstract Human dipeptidyl peptidase III (hDPP III), a zinc-metallopeptidase of the family M49, is an activator of the Keap1-Nrf2 cytoprotective pathway involved in defense against oxidative stress. Pathophysiological roles of DPP III have not been elucidated yet, partly due to the lack of specific inhibitors. We showed that substrate analog H-Tyr-Phe-NHOH is a strong competitive inhibitor of hDPP III, while H-Tyr-Gly-NHOH expresses much weaker inhibition. To investigate the effects of amino acid substitutions in inhibitor P1 position, we synthesized three new dipeptidyl hydroxamates and examined their influence on the activity of hDPP III and DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The extent of inhibition of hDPP III, but not of bacterial enzyme, was dependent on the amino acid in P1. H-Phe-Phe-NHOH is recognized as one of the strongest inhibitors of hDPP III (Ki = 0.028 μM), and H-Phe-Leu-NHOH discriminated between human and bacterial ortholog of the M49 family.

Hydrolysis of dipeptide derivatives reveals the diversity in the M49 family.

Abstract Dipeptidyl peptidase III, a metallopeptidase of the M49 family, was first identified (in the pituitary) by its specific cleavage of diarginyl arylamides, which have been used as preferred assay substrates until now. Here we examined the activity of the yeast and human dipeptidyl peptidase III in parallel. The human enzyme preferred Arg2-β-naphthylamide and showed 620-fold higher kcat/Km for this substrate. In contrast, the yeast enzyme did not display a preference for any of the X-Arg-β-naphthylamide analyzed. The replacement of Gly505 with Asp, resulted in a less active, but more selective, yeast enzyme form. These results indicate diversity in cleavage specificity in the M49 family.