X-Ray Absorption Spectroscopy of Dinuclear Metallohydrolases

Tierney, David L.; Schenk, Gerhard

doi:10.1016/j.bpj.2014.07.066

Cited by 18 publications

(23 citation statements)

References 99 publications

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“…EXAFS data collection and reduction were performed according to published procedures. 34 Data were measured in duplicate, six scans each on two independently-prepared samples. Fits to the two data sets were equivalent.…”

Section: Methodsmentioning

confidence: 99%

Probing substrate binding to the metal binding sites in metallo-β-lactamase L1 during catalysis

et al. 2016

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Metal ions in metallo-β-lactamases (MBLs) play a major role in catalysis. In this study we investigated the role of the metal ions in the Zn1 and Zn2 sites of MBL L1 during catalysis. A ZnCo (with Zn(II) in the invariant Zn1 site and Co(II) in the Zn2 site) analog of MBL L1 was prepared by using a biological incorporation method. Extended X-ray Absorption Fine Structure (EXAFS) spectroscopic studies were used to confirm that the ZnCo analog was prepared. To study the roles of the Zn(II) and Co(II) ions during catalysis, rapid freeze quench (RFQ)-EXAFS studies were used to probe the reaction of the ZnCo-L1 analog with chromacef when quenched at 10 ms, 50 ms, and 100 ms. The L1-product complex was also analyzed with EXAFS spectroscopy. The data show that the Zn-Co distance is 3.49 Å in the resting enzyme and that this distance increases by 0.3 Å in the sample that was quenched at 10 ms. The average Zn-Co distance decreases at the other time points until reaching a distance of 3.58 Å in the L1-product complex. The data also show that a Co-S interaction is present in the 100 ms quenched sample and in the L1-product complex, which suggests that there is a significant rearrangement of product in the active site.

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Section: Methodsmentioning

confidence: 99%

Probing substrate binding to the metal binding sites in metallo-β-lactamase L1 during catalysis

et al. 2016

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“…EXAFS samples were loaded in Lucite cuvettes with 6 µm polypropylene windows, flash-frozen and stored in liquid nitrogen. Freeze-quenched EXAFS samples were obtained using a modified Update Instruments (Madison, WI) rapid-freeze-quench (RFQ) system 55 . All enzyme and substrate starting concentrations were 1 mM, in 50 mM HEPES metal-free (with Chelex 100, Bio-Rad), pH 7.0, 20 % v/v glycerol.…”

Section: Methodsmentioning

confidence: 99%

A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases

et al. 2017

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Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-β-lactamases (MβLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MβLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MβLs. This common mechanism open avenues for rationally designed inhibitors of all MβLs, notwithstanding the profound differences between these enzymes’ active site structure, β-lactam specificity and metal content.

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“…We, and others, have explored extensively the functional roles model complexes can play in reproducing the electronic, structural and reactivity characteristics of organophosphatehydrolyzing metalloenzyme systems [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. One of the prominent issues targeted in these studies is the identity of the nucleophilic agent(s) in the models and in the corresponding metalloenzymes [27].…”

mentioning

confidence: 99%

Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by dinuclear mimics of metallohydrolases

Brown

Gahan

Schöffler

et al. 2016

Journal of Inorganic Biochemistry

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di-Zinc(II) complexes of the ligands 2,6-bis((bis(2-methoxyethyl)amino)methyl)-4-methylphenol (HL1), 2,6-bis(bis(hydroxyethyl)aminomethyl)-4-methylphenol (HL2) and 2,6-bis((hydroxyethyl)(methoxyethyl)-aminomethyl)-4-methylphenol (HL3) have been prepared and characterized. The three ligands differ in their donor types, having ether donors (HL1), alkoxido donors (HL2) and both ether and alkoxido donors (HL3). These differences allowed an investigation into the role of the potential nucleophiles in the hydrolysis reaction with the phosphodiester substrate bis(2,4-dinitrophenyl)phosphate (BDNPP). In addition, the di-Mg(II) complex of ligand HL2 was prepared in order to examine the potential for Mg(II) to replace Zn(II) in these biomimetic systems. Kinetically relevant pK values for the three di-Zn(II) complexes were determined to be 7.14 and 9.21 for [Zn(L1)(CHCOO)](PF), 7.90 and 10.21 for [Zn(L2)(CHCOO)](BPh) and 8.43 and 10.69 for [Zn(L3)(CHCOO)](BPh). At the respective pH optima the relevant catalytic parameters are k=5.44(0.11)×10s (K=5.13(0.92) mM), 2.60(0.87)×10s (K=5.49(1.51) mM) and 1.53(0.27)×10s (K=2.14(0.50) mM) for [Zn(L1)(CHCOO)](PF), [Zn(L2)(CHCOO)](BPh) or [Zn(L3)(CHCOO)](BPh), respectively. The di-Mg(II) complex was found to be unreactive in the hydrolysis reaction with BDNPP under the conditions employed. Computational methods using the [Zn(L2)(CHCOO)](BPh) complex were used to discriminate between different possible mechanistic pathways. The DFT calculations indicate that an alkoxido-mediated pathway in the complexes formed with ligands L2 or L3 is unlikely, because it induces significant distortion of the Zn(L) unit; a direct attack by a coordinated hydroxide is preferred in each of the three systems studied here. The calculations also revealed the important role of ligand structural rigidity.

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X-Ray Absorption Spectroscopy of Dinuclear Metallohydrolases

Cited by 18 publications

References 99 publications

Probing substrate binding to the metal binding sites in metallo-β-lactamase L1 during catalysis

Probing substrate binding to the metal binding sites in metallo-β-lactamase L1 during catalysis

A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases

Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by dinuclear mimics of metallohydrolases

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