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

Špoljarić, Jasminka; Salopek‐Sondi, Branka; Makarević, Janja; Vukelić, Bojana; Agić, Dejan; Šimaga, Šumski; Jajčanin-Jozić, Nina; Abramić, Marija

doi:10.1016/j.bioorg.2009.03.002

Cited by 27 publications

(42 citation statements)

References 22 publications

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“…The competitive character of this inhibition was demonstrated by Dixon and Cornish-Bowden (s/v versus i) plots 20 .…”

Section: Effects Of Synthesized Dipeptidyl Hydroxamic Acids On the Acmentioning

confidence: 82%

“…H-Tyr-Phe-NHOH inhibited hDPP III with the K i value of 0.15 mM at pH 8.0 20 . However, we observed a dramatic decrease in the inhibitory potency of H-Tyr-Gly-NHOH (70-fold higher K i value), indicating that for the strong interactions of DPP III with hydroxamate inhibitor, chelating effect of hydroxamate moiety is not sufficient, but, in addition, interactions between the amino acid side chains of dipeptidyl hidroxamate with substrate binding subsites S1 and S2 are of crucial importance.…”

Section: Introductionmentioning

confidence: 93%

“…Enzyme inhibition studies were performed using the Arg 2 -2NA as substrate. Dixon and s/v versus i plots were used to establish the type of inhibition exhibited by hydroxamate inhbitor at pH 8.0 and to determine the K i values 20 . At pH 7.4, the inhibition constant, K i , was calculated according to the equation:…”

Section: Determination Of Kinetic Parametersmentioning

confidence: 99%

“…Although a significant knowledge on the molecular enzymology (structure-activity relationship) of the DPP III family accumulated in the last decade [19][20][21][22] , physiological roles of any of its members have not been elucidated in sufficient detail. This is partly due to the lack of specific inhibitors.…”

Section: Introductionmentioning

confidence: 99%

“…Inhibition by nonspecific chelating and thiol reagents is a characteristics of DPP III from various species 2,16,23 , but the studies of more specific inhibitors of the DPP III enzymatic activity are rare 24 . We have shown that two dipeptidyl hydroxamic acids, H-Tyr-Phe-NHOH and H-Tyr-Gly-NHOH act as the competitive inhibitors of human DPP III activity 20 . Hydroxamate moiety (-NHOH) is capable of chelating the zinc ion at the catalytic site, and therefore it is essential for DPP III inhibition by dipeptidyl hydroxamic acids.…”

Section: Introductionmentioning

confidence: 99%

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

Cvitešić¹,

Sabljić

Makarević³

et al. 2016

Journal of Enzyme Inhibition and Medicinal Chemistry

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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 (K i ¼ 0.028 mM), and H-Phe-Leu-NHOH discriminated between human and bacterial ortholog of the M49 family.

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“…The competitive character of this inhibition was demonstrated by Dixon and Cornish-Bowden (s/v versus i) plots 20 .…”

Section: Effects Of Synthesized Dipeptidyl Hydroxamic Acids On the Acmentioning

confidence: 82%

Section: Introductionmentioning

confidence: 93%

Section: Determination Of Kinetic Parametersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Cvitešić¹,

Sabljić

Makarević³

et al. 2016

Journal of Enzyme Inhibition and Medicinal Chemistry

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

Tomić

Abramić

Špoljarić

et al. 2011

J of Molecular Recognition

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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.

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Dipeptidyl-Peptidase III

Barrett

2013

Handbook of Proteolytic Enzymes

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

Cited by 27 publications

References 22 publications

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

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

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

Dipeptidyl-Peptidase III

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