Binding of hydroxamic acid inhibitors to crystalline thermolysin suggests a pentacoordinate zinc intermediate in catalysis

Holmes, Michael; Matthews, B.W.

doi:10.1021/bi00527a026

Cited by 236 publications

(127 citation statements)

References 36 publications

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“…A similar involvement of Glul43 in the binding of HONH-isobutylmalonyl-Ala-Gly-NH, to thermolysin was established from kinetic measurements (Izquierdo-Martin and Stein, 1992). Importantly, in all the previously reported structures of metallopeptidases complexed with a hydroxamate inhibitor, the interactions of an acidic glutamic acid via its carboxyl oxygens with the hydroxylamino nitrogen and oxygen appears as a general common feature (Holmes and Matthews, 1981;Borkakoti et al, 1994;Spurlino et al, 1994;Bode et al, 1994;Stams et al, 1994). The conservation of a glutamic acid residue in the active site suggests that the hydrolytic mechanism of these metalloproteases could be closely related.…”

Section: Discussionsupporting

confidence: 66%

The Structure of the Aeromonas proteolytica Aminopeptidase Complexed with a Hydroxamate Inhibitor

Chevrier

d'Orchymont²,

Schalk³

et al. 1996

European Journal of Biochemistry

139

134

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The structure of the complex of Aeromonas proteolytica aminopeptidase, a two-zinc exopeptidase, with the inhibitor p-iodo-D-phenylalanine hydroxarnate has been determined by X-ray crystallography. Refinement of the structure, which includes 220 water molecules, using data at 0.80-0.23-nm resolution resulted in a crystallographic residual R value of 16%. The hydroxamate group adopts a planar conformation whereby the two oxygen atoms interact with the zinc ions. The N-hydroxyl group of the inhibitor is located between the two zinc ions, a position which is close to that occupied by a water molecule in the native structure. The carbonyl oxygen of the inhibitor binds to Znl, which becomes pentacoordinated while Zn2 remains tetracoordinated, in contrast to the native protein where both zinc ions were shown to be tetracoordinated and structurally equivalent. Interactions of the carboxylate oxygens of Glu151 with the hydroxamate group play an important role in the stabilization of the complex.

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Section: Discussionsupporting

confidence: 66%

The Structure of the Aeromonas proteolytica Aminopeptidase Complexed with a Hydroxamate Inhibitor

Chevrier

d'Orchymont²,

Schalk³

et al. 1996

European Journal of Biochemistry

139

134

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“…Initially, monodentate complex with inhibitor is formed with displacement of zinc-bound water molecule, and formation of stable bidentate complex is accompanied by ionization of the bound hydroxamic acid. Earlier, the mode of binding of a series of hydroxamic acid derivatives, potent inhibitors of thermolysin has been determined by X-ray crystallography 35 , revealing that hydroxamate moiety forms a bidentate complex with the zinc. For another metallopeptidase, carboxypeptidase A (CP A), graphical presentation of the pH dependence of pK i for substrate analog hydroxamic acids derivatives was a curve which exhibits a maximum near neutrality 32 .…”

Section: Effects Of Synthesized Dipeptidyl Hydroxamic Acids On the Acmentioning

confidence: 99%

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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“…In the latter, one of the two active-site zinc ions is coordinated both by the side-chain carboxylate and the main-chain carbonyl group of an aspartic acid residue. Additional carbonyl -zinc interactions are found in several complexes with peptide inhibitors of the hydrolytic enzymes carboxypeptidase A (Christiansson and Lipscomb, 1986) and thermolysin (Holmes and Matthews, 1981;Holmes et al, 1983).…”

Section: Z I N C Binding To Azurinmentioning

confidence: 99%

Characterization and crystal structure of zinc azurin, a by‐product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin

Nar¹,

Huber²,

Messerschmidt³

et al. 1992

European Journal of Biochemistry

147

151

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Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copperions.An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45.The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, whch is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.

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Binding of hydroxamic acid inhibitors to crystalline thermolysin suggests a pentacoordinate zinc intermediate in catalysis

Cited by 236 publications

References 36 publications

The Structure of the Aeromonas proteolytica Aminopeptidase Complexed with a Hydroxamate Inhibitor

The Structure of the Aeromonas proteolytica Aminopeptidase Complexed with a Hydroxamate Inhibitor

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

Characterization and crystal structure of zinc azurin, a by‐product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin

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