Metal ion promoted phosphate ester hydrolysis. Intramolecular attack of coordinated hydroxide ion

Hendry, Philip; Sargeson, Alan M.

doi:10.1021/ja00189a025

Cited by 111 publications

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“…Ethyl para-nitrophenyl phosphate (EtpNPP) was synthesized by known methods. 27 Chemical structures of the substrates used in this study are shown in Figure S1.…”

Section: Methodsmentioning

confidence: 99%

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

et al. 2009

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Abstract:The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K d values for the metal ions in the R and sites to be 29 and 344 µM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the site increases substantially (K d ) 56 µM), while that of the R site is not greatly affected (K d ) 17 µM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k obs1 ), the assembly of a catalytically active binuclear center (k obs2 ), and subsequent slower structural rearrangements to optimize catalysis (k obs3 ). These three phases depend on the concentration of substrate ([S]), with k obs1 and k obs2 reaching maximum values at high [S] (354 and 38 s -1 , respectively), whereas k obs3 is reduced as [S] is increased. The k cat for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (∼1 s -1 ) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k obs3 is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate-and metal-ion-induced fit to optimize catalysis.

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“…Ethyl para-nitrophenyl phosphate (EtpNPP) was synthesized by known methods. 27 Chemical structures of the substrates used in this study are shown in Figure S1.…”

Section: Methodsmentioning

confidence: 99%

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

et al. 2009

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“…Metal cation-nucleic acid interactions are currently of great interest because metal cations and metal cation-ligand complexes bind to DNA and regulate gene expression, act as drugs, or can be used as tools for molecular biology studies [8]. Hendry and Sargeson studied metal cation-promoted phosphate ester hydrolysis and found that the rate of hydrolysis is modulated by the size of the metal cation and the ease of formation of a four-membered chelate ring [9]. Dempcy and Bruice found a pentacoordinate intermediate for the catalysis of alkyl phosphate diesters [10].…”

mentioning

confidence: 99%

A simple model for metal cation-phosphate interactions in nucleic acids in the gas phase: Alkali metal cations and trimethyl phosphate

Ruan

Huang

Rodgers

2008

J. Am. Soc. Mass Spectrom.

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Threshold collision-induced dissociation techniques are employed to determine the bond dissociation energies (BDEs) of complexes of alkali metal cations to trimethyl phosphate, TMP. Endothermic loss of the intact TMP ligand is the only dissociation pathway observed for all complexes. Theoretical calculations at the B3LYP/6-31G* level of theory are used to determine the structures, vibrational frequencies, and rotational constants of neutral TMP and the M ϩ (TMP) complexes. Theoretical BDEs are determined from single point energy calculations at the B3LYP/6-311ϩG(2d,2p) level using the B3LYP/6-31G* optimized geometries. The agreement between theory and experiment is reasonably good for all complexes except Li ϩ (TMP hosphate esters are integral parts of many biologically active molecules ranging from nucleic acids, lipids, and ATP to pesticides and nerve agents [1][2][3]. Serving as link components in nucleic acids, phosphate esters connect thousands of base pairs to form large molecules that contain the genetic information. These phosphate groups are deprotonated under physiological conditions. The resulting negative charges along the phosphate backbone are stabilized and neutralized by the binding of metal cations. Metal cations can and often do exert a significant influence on nucleic acid structure and genetic information transfer [4]. Metal cation binding reduces the repulsion between neighboring nucleotide units, and therefore influences the stabilization and structural dynamics of nucleic acids and lipids in membranes. For example, alkali and alkaline earth metal cations, including the most biologically important of these cations, Na ϩ and Mg 2ϩ , prefer to bind to a phosphate group rather than to the nucleobases or sugar moieties [5]. Mg 2ϩ is found to increase the melting temperature of DNA [6]. Metal cations may also be involved in the functioning of nucleic acids, e.g., metal cations are required for the proper functioning of all ribozymes [7]. Metal cation-nucleic acid interactions are currently of great interest because metal cations and metal cation-ligand complexes bind to DNA and regulate gene expression, act as drugs, or can be used as tools for molecular biology studies [8]. Hendry and Sargeson studied metal cation-promoted phosphate ester hydrolysis and found that the rate of hydrolysis is modulated by the size of the metal cation and the ease of formation of a four-membered chelate ring [9]. Dempcy and Bruice found a pentacoordinate intermediate for the catalysis of alkyl phosphate diesters [10]. Schneider and Kabelac et al. [11] studied the distribution of five metal cations (i.e., Na, and Zn 2ϩ ) and water around a negatively charged phosphate group from various crystal structures, and found that all of the cations prefer asymmetric monodentate coordination over bidentate coordination. Their ab initio calculations suggest that bidentate binding of these metal cations to an isolated phosphate group is preferred, but that the presence of even a single water molecule alters this preference. Ab...

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“…The exceptional chemical stability of the phosphodiester backbone in molecules such as RNA and DNA facilitates the conservation of genetic information. An array of model compounds that promote the catalytic hydrolysis of phosphodiester bonds has been designed and synthesized (17)(18)(19)(20)(21)(22)(23). Nearly all of the model compounds constructed to date utilize metal ions as essential cofactors to activate the substrate and nucleophile while holding the reactants together in close proximity (17-21).…”

mentioning

confidence: 99%

“…For example, it has been estimated that ethyl-4-nitrophenyl phosphate (Structure 1, compound I) spontaneously hydrolyzes with k obs Յ 10 Ϫ10 s Ϫ1 at 25°C and pH 8 (17), whereas under the same reaction conditions, the base-catalyzed hydrolysis of paraoxon, diethyl-4-nitrophenyl phosphate (Structure 1, compound II), proceeds with a rate constant of ϳ10 Ϫ7 s Ϫ1 (1). The exceptional chemical stability of the phosphodiester backbone in molecules such as RNA and DNA facilitates the conservation of genetic information.…”

mentioning

confidence: 99%

Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase

Shim

Hong

Raushel

1998

Journal of Biological Chemistry

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The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10 8 times faster than the uncatalyzed reaction (k cat ‫؍‬ 0.06 s ؊1 and K m ‫؍‬ 38 mM). Upon the addition of various alkylamines to the reaction mixture, the k cat /K m for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (k cat ‫؍‬ 0.34 s ؊1 and K m ‫؍‬ 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wildtype enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide range of organophosphate nerve agents with high efficiency (1, 2). Paraoxon, the best characterized substrate for the zinc-substituted phosphotriesterase, is hydrolyzed with a k cat /K m of 4 ϫ 10and a k cat of 2100 s Ϫ1 . The active site of this enzyme contains a coupled binuclear metal center, which is absolutely essential for catalytic activity (1). The native enzyme contains two Zn 2ϩ ions, but these metal ions can be replaced with Co 2ϩ , Ni 2ϩ, Mn 2ϩ , or Cd 2ϩ with retention of full catalytic activity. From chemical, kinetic, and genetic studies, it has been demonstrated that the reaction proceeds via an S n 2-like associative mechanism in which a metal-bound hydroxide ion attacks the electrophilic phosphorus center of the substrate (2-16). The role of one of the two metal ions within the active site is thought to involve the activation of the hydrolytic water molecule, whereas the companion metal ion is most likely involved in the polarization of the phosphoryl oxygen bond of the substrate to increase the electrophilicity of the substrate for nucleophilic attack (16). Not surprisingly, the substrate-binding site pocket consists predominantly of ...

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Metal ion promoted phosphate ester hydrolysis. Intramolecular attack of coordinated hydroxide ion

Cited by 111 publications

References 1 publication

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

A simple model for metal cation-phosphate interactions in nucleic acids in the gas phase: Alkali metal cations and trimethyl phosphate

Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase

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