A Structural and Functional Model of Dinuclear Metallophosphatases

Williams, Nicholas H.; Lebuis, and Anne-Marie; Chin, Jik

doi:10.1021/ja9827797

Cited by 85 publications

(79 citation statements)

References 40 publications

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“…Alternatively, if the diester binds in a bidentate fashion, attack by the bridging hydroxide would produce a tripodally coordinated intermediate, which could rapidly add water from solvent to yield a bridging monoester complex (precedent for such a process comes from the reactions of phosphodiesters bound to a di-Co 3+ complex). 12 While the second possibility cannot be ruled out, we propose that the first alternative is more consistent with the kinetic data and with previous observations. Phosphodiesters bind and undergo hydrolysis by PAP model systems in the manner we propose for the enzymatic reaction.…”

supporting

confidence: 87%

Diesterase Activity and Substrate Binding in Purple Acid Phosphatases

et al. 2007

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Purple acid phosphatases (PAPs) are dinuclear monoesterases 1,2 that are structurally diverse but with a conserved set of ligands to the Fe 3+ -M 2+ metal centers (M 2+ ) Fe, Zn, or Mn). 3 The identity of the nucleophile and the substrate binding mode have been matters of controversy (Figure 1). 1,[3][4][5] In one proposal, substrate binds to the divalent metal and is attacked by a terminal Fe 3+ -bound hydroxide. In an alternative mechanism, substrate coordinates first to the divalent metal and then forms a bridging complex, followed by nucleophilic attack by the µ-hydroxide. We report that the PAPs from pig and kidney bean catalyze the hydrolysis of diesters if the second ester group is small, and the kinetics of the reaction with methyl p-nitrophenylphosphate suggest that a terminal-bound hydroxide is the nucleophile for the diester, followed by attack of the bridging hydroxide upon the resulting monoester without release into solution.The PAPs are reported to lack the ability to hydrolyze diesters, based on the lack of activity with bis(p-nitrophenyl)phosphate (BpNPP). 6 In contrast, model complexes designed to mimic the PAP metal center hydrolyze diesters, while monoesterase activity has not been reported. 7 This suggests that the inability of PAP to hydrolyze BpNPP might result from steric considerations. Thus, we tested the PAPs from pig (also termed uteroferrin) and from red kidney bean using two diesters bearing smaller second ester groups, methyl pNPP (MpNPP) and ethyl pNPP (EpNPP). While BpNPP gave negligible turnover, consistent with a previous report, 6 the other two diesters, particularly MpNPP, are substrates although binding is poor. The pH dependence for k cat and k cat /K m of the reactions with MpNPP was followed by monitoring release of p-nitrophenol (see Supporting Information, Figure S1). The pH optima for monoesterase and diesterase reactions were the same; data in Table 1 were obtained at the optimal pH of 4.9 for pig PAP and pH 6.25 for kbPAP (see Supporting Information for EpNPP and BpNPP data).At its pH optimum, the k cat for MpNPP hydrolysis by pig PAP is close to that for the monoester pNPP, 5,8 while for kbPAP, the MpNPP k cat is about half that for pNPP.The high diester K m values presumably result from steric requirements of the second ester group. 14 For comparison, K m values of aryl and benzyl monoesters range from 1.25 to 5.8 mM. 5 Inorganic phosphate is a competitive inhibitor of the reactions of both PAPs with MpNPP, with K i values close to the literature values for the reactions with pNPP ( Table 2).The reaction of MpNPP is assumed to first yield p-nitrophenol and methyl phosphate, on the basis of the large difference in the pK a values of the two potential leaving groups and the observation that PAP catalysis shows a significant dependence on the leaving group ( lg ) -0.4 and -0.6 for pig and kbPAP, respectively). 5 Methyl phosphate (MP), the presumed product of the diester reaction, is a substrate, with k cat values of 17 and 20 s -1 for the pig and kbPAPs respectively,...

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confidence: 87%

Diesterase Activity and Substrate Binding in Purple Acid Phosphatases

et al. 2007

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“…Upon substrate binding, we predict that the bridging hydroxide becomes terminally bound to Zn 2 while retaining a hydrogen bond to Asp-120. As predicted with model complexes (61), other Zn(II)-and Fe-containing proteins (62)(63)(64)(65), computational studies on metallo-␤-lactamases (40,42,66), and with other metallo-␤-lactamases (57, 67, 68), this metal bound hydroxide then serves as the nucleophile that attacks the ␤-lactam carbonyl. The kinetic data clearly show that D120N is the only mutant that retains significant hydrolytic activity.…”

Section: Asp-120 Mutants Of Metallo-␤-lactamase L1mentioning

confidence: 60%

Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis

Garrity

Carenbauer

Herron

et al. 2004

Journal of Biological Chemistry

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Metallo-␤-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a ␤-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 ؎ 0.2, 1.8 ؎ 0.2, and 1.1 ؎ 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10-to 1000-fold drops in k cat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pK a values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.

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“…In contrast, the lack of a dependence of its kinetic parameters on the leaving group pK a at pH 4.90 may indicate that sweet potato PAP has an associative transition state; i.e., bond formation by the nucleophile is more significant than bond breaking to release the leaving group. In this respect the mechanism used by sweet potato PAP resembles that proposed for a binuclear Co(III) complex shown by kinetic isotope effects to use the bridging oxo group as nucleophile (70,71). It was suggested that the transition state displays a considerable degree of bond formation as evidenced by the modest charge development on the leaving group (70).…”

Section: Mechanistic Implications For Sweet Potato Papmentioning

confidence: 81%