Raushel FM


Full name : Raushel Frank M

First name : Frank M

Mail : Department of Chemistry, Texas AM University, College Station, TX 77843-3255

Zip Code :

City :

Country : USA

Email : raushel@tamu.edu

Phone : (1-409) 8453373

Fax : (1-409) 8459452

Website :

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References (29)

Title : Substrate Analogues for the Enzyme-Catalyzed Detoxification of the Organophosphate Nerve Agents-Sarin, Soman, and Cyclosarin - Bigley_2021_Biochemistry__
Author(s) : Bigley AN , Harvey SP , Narindoshvili T , Raushel FM
Ref : Biochemistry , : , 2021
Abstract : The G-type nerve agents, sarin (GB), soman (GD), and cyclosarin (GF), are among the most toxic compounds known. Much progress has been made in evolving the enzyme phosphotriesterase (PTE) from Pseudomonas diminuta for the decontamination of the G-agents; however, the extreme toxicity of the G-agents makes the use of substrate analogues necessary. Typical analogues utilize a chromogenic leaving group to facilitate high-throughput screening, and substitution of an O-methyl for the P-methyl group found in the G-agents, in an effort to reduce toxicity. Till date, there has been no systematic evaluation of the effects of these substitutions on catalytic activity, and the presumed reduction in toxicity has not been tested. A series of 21 G-agent analogues, including all combinations of O-methyl, p-nitrophenyl, and thiophosphate substitutions, have been synthesized and evaluated for their ability to unveil the stereoselectivity and catalytic activity of PTE variants against the authentic G-type nerve agents. The potential toxicity of these analogues was evaluated by measuring the rate of inactivation of acetylcholinesterase (AChE). All of the substitutions reduced inactivation of AChE by more than 100-fold, with the most effective being the thiophosphate analogues, which reduced the rate of inactivation by about 4-5 orders of magnitude. The analogues were found to reliably predict changes in catalytic activity and stereoselectivity of the PTE variants and led to the identification of the BHR-30 variant, which has no apparent stereoselectivity against GD and a k(cat)/K(m) of 1.4 x 10(6), making it the most efficient enzyme for GD decontamination reported till date.
ESTHER : Bigley_2021_Biochemistry__
PubMedSearch : Bigley_2021_Biochemistry__
PubMedID: 34494832

Title : The evolution of phosphotriesterase for decontamination and detoxification of organophosphorus chemical warfare agents - Bigley_2019_Chem.Biol.Interact_13ChEPon_308_80
Author(s) : Bigley AN , Raushel FM
Ref : Chemico-Biological Interactions , 308 :80 , 2019
Abstract : The organophosphorus chemical warfare agents were initially synthesized in the 1930's and are some of the most toxic compounds ever discovered. The standard means of decontamination are either harsh chemical hydrolysis or high temperature incineration. Given the continued use of chemical warfare agents there are ongoing efforts to develop gentle environmentally friendly means of decontamination and medical counter measures to chemical warfare agent intoxication. Enzymatic decontamination offers the benefits of extreme specificity and mild conditions, allowing their use for both environmental and medical applications. The most promising enzyme for decontamination of the organophosphorus chemical warfare agents is the enzyme phosphotriesterase from Pseudomonas diminuta. However, the catalytic activity of the wild-type enzyme with the chemical warfare agents falls far below that seen with its best substrates, and its stereochemical preference is for the less toxic enantiomer of the chiral phosphorus center found in most chemical warfare agents. Rational design efforts have succeeded in the dramatic improvement of the stereochemical preference of PTE for the more toxic enantiomers. Directed evolution experiments, including site-saturation mutagenesis, targeted error-prone PCR, computational design, and quantitative library analysis, have systematically improved the catalytic activity against the chemical warfare nerve agents. These efforts have resulted in greater than 4-orders of magnitude improvement in catalytic activity and have led to the identification of variants that are highly effective at detoxifying both G-type and V-type nerve agents. The best of these variants have the ability to prevent intoxication when delivered as a post-exposure treatment for VX and as a pre-exposure treatment for G-agent intoxication with observed protective factors up to 60-fold. Combining the best variant, H257Y/L303T, with a PCB polymer coating has enabled the development of a long lasting circulating prophylactic treatment that is highly effective against sarin.
ESTHER : Bigley_2019_Chem.Biol.Interact_13ChEPon_308_80
PubMedSearch : Bigley_2019_Chem.Biol.Interact_13ChEPon_308_80
PubMedID: 31100274

Title : An OPAA enzyme mutant with increased catalytic efficiency on the nerve agents sarin, soman, and GP - Bae_2018_Enzyme.Microb.Technol_112_65
Author(s) : Bae SY , Myslinski JM , McMahon LR , Height JJ , Bigley AN , Raushel FM , Harvey SP
Ref : Enzyme Microb Technol , 112 :65 , 2018
Abstract : The wild-type OPAA enzyme has relatively high levels of catalytic activity against several organophosphate G-type nerve agents. A series of mutants containing replacement amino acids at the OPAA Y212, V342, and I215 sites showed several fold enhanced catalytic efficiency on sarin, soman, and GP. One mutant, Y212F/V342L, showed enhanced stereospecificity on sarin and that enzyme along with a phosphotriesterase mutant, GWT, which had the opposite stereospecificity, were used to generate enriched preparations of each sarin enantiomer. Inhibition of acetylcholinesterase by the respective enantioenriched sarin solutions subsequently provided identification of the sarin enantiomers as separated by normal phase enantioselective liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry.
ESTHER : Bae_2018_Enzyme.Microb.Technol_112_65
PubMedSearch : Bae_2018_Enzyme.Microb.Technol_112_65
PubMedID: 29499783

Title : Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from Sphingobium sp. TCM1 - Bigley_2018_Biochemistry_57_1842
Author(s) : Bigley AN , Narindoshvili T , Xiang DF , Raushel FM
Ref : Biochemistry , 57 :1842 , 2018
Abstract : The phosphotriesterase from Sphingobium sp. TCM1 ( Sb-PTE) is notable for its ability to hydrolyze organophosphates that are not substrates for other enzymes. In an attempt to determine the catalytic properties of Sb-PTE for hydrolysis of chiral phosphotriesters, we discovered that multiple phosphodiester products are formed from a single substrate. For example, Sb-PTE catalyzes the hydrolysis of the RP-enantiomer of methyl cyclohexyl p-nitrophenyl phosphate with exclusive formation of methyl cyclohexyl phosphate. However, the enzyme catalyzes hydrolysis of the SP-enantiomer of this substrate to an equal mixture of methyl cyclohexyl phosphate and cyclohexyl p-nitrophenyl phosphate products. The ability of this enzyme to catalyze the hydrolysis of a methyl ester at the same rate as the hydrolysis of a p-nitrophenyl ester contained within the same substrate is remarkable. The overall scope of the stereoselective properties of this enzyme is addressed with a library of chiral and prochiral substrates.
ESTHER : Bigley_2018_Biochemistry_57_1842
PubMedSearch : Bigley_2018_Biochemistry_57_1842
PubMedID: 29513982

Title : Interrogation of the Substrate Profile and Catalytic Properties of the Phosphotriesterase from Sphingobium sp. Strain TCM1: An Enzyme Capable of Hydrolyzing Organophosphate Flame Retardants and Plasticizers - Xiang_2015_Biochemistry_54_7539
Author(s) : Xiang DF , Bigley AN , Ren Z , Xue H , Hull KG , Romo D , Raushel FM
Ref : Biochemistry , 54 :7539 , 2015
Abstract : The most familiar organophosphorus compounds are the neurotoxic insecticides and nerve agents. A related group of organophosphorus compounds, the phosphotriester plasticizers and flame retardants, has recently become widely used. Unlike the neurotoxic phosphotriesters, the plasticizers and flame retardants lack an easily hydrolyzable bond. While the hydrolysis of the neurotoxic organophosphates by phosphotriesterase enzymes is well-known, the lack of a labile bond in the flame retardants and plasticizers renders them inert to typical phosphotriesterases. A phosphotriesterase from Sphingobium sp. strain TCM1 (Sb-PTE) has recently been reported to catalyze the hydrolysis of organophosphorus flame retardants. This enzyme has now been expressed in Escherichia coli, and the activity with a wide variety of organophosphorus substrates has been characterized and compared to the activity of the well-known phosphotriesterase from Pseudomonas diminuta (Pd-PTE). Structure prediction suggests that Sb-PTE has a beta-propeller fold, and homology modeling has identified a potential mononuclear manganese binding site. Sb-PTE exhibits catalytic activity against typical phosphotriesterase substrates such as paraoxon, but unlike Pd-PTE, Sb-PTE is also able to effectively hydrolyze flame retardants, plasticizers, and industrial solvents. Sb-PTE can hydrolyze both phosphorus-oxygen bonds and phosphorus-sulfur bonds, but not phosphorus-nitrogen bonds. The best substrate for Sb-PTE is the flame retardant triphenyl phosphate with a kcat/Km of 1.7 x 10(6) M(-1) s(-1). Quite remarkably, Sb-PTE is also able to hydrolyze phosphotriesters with simple alcohol leaving groups such as tributyl phosphate (kcat/Km = 40 M(-1) s(-1)), suggesting that this enzyme could be useful for the bioremediation of a wide variety of organophosphorus compounds.
ESTHER : Xiang_2015_Biochemistry_54_7539
PubMedSearch : Xiang_2015_Biochemistry_54_7539
PubMedID: 26629649

Title : Catalytic mechanisms for phosphotriesterases - Bigley_2013_Biochim.Biophys.Acta_1834_443
Author(s) : Bigley AN , Raushel FM
Ref : Biochimica & Biophysica Acta , 1834 :443 , 2013
Abstract : Phosphotriesters are one class of highly toxic synthetic compounds known as organophosphates. Wide spread usage of organophosphates as insecticides as well as nerve agents has lead to numerous efforts to identify enzymes capable of detoxifying them. A wide array of enzymes has been found to have phosphotriesterase activity including phosphotriesterase (PTE), methyl parathion hydrolase (MPH), organophosphorus acid anhydrolase (OPAA), diisopropylfluorophosphatase (DFP), and paraoxonase 1 (PON1). These enzymes differ widely in protein sequence and three-dimensional structure, as well as in catalytic mechanism, but they also share several common features. All of the enzymes identified as phosphotriesterases are metal-dependent hydrolases that contain a hydrophobic active site with three discrete binding pockets to accommodate the substrate ester groups. Activation of the substrate phosphorus center is achieved by a direct interaction between the phosphoryl oxygen and a divalent metal in the active site. The mechanistic details of the hydrolytic reaction differ among the various enzymes with both direct attack of a hydroxide as well as covalent catalysis being found. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.
ESTHER : Bigley_2013_Biochim.Biophys.Acta_1834_443
PubMedSearch : Bigley_2013_Biochim.Biophys.Acta_1834_443
PubMedID: 22561533

Title : Molecular engineering of organophosphate hydrolysis activity from a weak promiscuous lactonase template - Meier_2013_J.Am.Chem.Soc_135_11670
Author(s) : Meier MM , Rajendran C , Malisi C , Fox NG , Xu C , Schlee S , Barondeau DP , Hocker B , Sterner R , Raushel FM
Ref : Journal of the American Chemical Society , 135 :11670 , 2013
Abstract : Rapid evolution of enzymes provides unique molecular insights into the remarkable adaptability of proteins and helps to elucidate the relationship between amino acid sequence, structure, and function. We interrogated the evolution of the phosphotriesterase from Pseudomonas diminuta (PdPTE), which hydrolyzes synthetic organophosphates with remarkable catalytic efficiency. PTE is thought to be an evolutionarily 'young' enzyme, and it has been postulated that it has evolved from members of the phosphotriesterase-like lactonase (PLL) family that show promiscuous organophosphate-degrading activity. Starting from a weakly promiscuous PLL scaffold (Dr0930 from Deinococcus radiodurans ), we designed an extremely efficient organophosphate hydrolase (OPH) with broad substrate specificity using rational and random mutagenesis in combination with in vitro activity screening. The OPH activity for seven organophosphate substrates was simultaneously enhanced by up to 5 orders of magnitude, achieving absolute values of catalytic efficiencies up to 10(6) M(-1) s(-1). Structural and computational analyses identified the molecular basis for the enhanced OPH activity of the engineered PLL variants and demonstrated that OPH catalysis in PdPTE and the engineered PLL differ significantly in the mode of substrate binding.
ESTHER : Meier_2013_J.Am.Chem.Soc_135_11670
PubMedSearch : Meier_2013_J.Am.Chem.Soc_135_11670
PubMedID: 23837603

Title : Enzymes for the homeland defense: optimizing phosphotriesterase for the hydrolysis of organophosphate nerve agents - Tsai_2012_Biochemistry_51_6463
Author(s) : Tsai PC , Fox N , Bigley AN , Harvey SP , Barondeau DP , Raushel FM
Ref : Biochemistry , 51 :6463 , 2012
Abstract : Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center, and the S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of the wild-type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 x 10(6), 5 x 10(5), and 8 x 10(5) M(-1) s(-1), respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.
ESTHER : Tsai_2012_Biochemistry_51_6463
PubMedSearch : Tsai_2012_Biochemistry_51_6463
PubMedID: 22809162

Title : Stereoselective hydrolysis of organophosphate nerve agents by the bacterial phosphotriesterase - Tsai_2010_Biochemistry_49_7978
Author(s) : Tsai PC , Bigley A , Li Y , Ghanem E , Cadieux CL , Kasten SA , Reeves TE , Cerasoli DM , Raushel FM
Ref : Biochemistry , 49 :7978 , 2010
Abstract : Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well-known are GA, GB, GD, GF, VX, and VR. All of these compounds contain a chiral phosphorus center, with the S(P) enantiomers being significantly more toxic than the R(P) enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R(P) enantiomers. A series of enantiomerically pure chiral nerve agent analogues containing the relevant phosphoryl centers found in GB, GD, GF, VX, and VR has been developed. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed, stereoselectivity have been identified. A number of variants exhibited dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S(P) enantiomers. Improvements of up to 3 orders of magnitude relative to the value of the wild-type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.
ESTHER : Tsai_2010_Biochemistry_49_7978
PubMedSearch : Tsai_2010_Biochemistry_49_7978
PubMedID: 20701311

Title : Structure of diethyl phosphate bound to the binuclear metal center of phosphotriesterase - Kim_2008_Biochemistry_47_9497
Author(s) : Kim J , Tsai PC , Chen SL , Himo F , Almo SC , Raushel FM
Ref : Biochemistry , 47 :9497 , 2008
Abstract : The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters at rates close to the diffusion limit. X-ray diffraction studies have shown that a binuclear metal center is positioned in the active site of PTE and that this complex is responsible for the activation of the nucleophilic water from solvent. In this paper, the three-dimensional structure of PTE was determined in the presence of the hydrolysis product, diethyl phosphate (DEP), and a product analogue, cacodylate. In the structure of the PTE-diethyl phosphate complex, the DEP product is found symmetrically bridging the two divalent cations. The DEP displaces the hydroxide from solvent that normally bridges the two divalent cations in structures determined in the presence or absence of substrate analogues. One of the phosphoryl oxygen atoms in the PTE-DEP complex is 2.0 A from the alpha-metal ion, while the other oxygen is 2.2 A from the beta-metal ion. The two metal ions are separated by a distance of 4.0 A. A similar structure is observed in the presence of cacodylate. Analogous complexes have previously been observed for the product complexes of isoaspartyl dipeptidase, d-aminoacylase, and dihydroorotase from the amidohydrolase superfamily of enzymes. The experimentally determined structure of the PTE-diethyl phosphate product complex is inconsistent with a recent proposal based upon quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the beta-metal ion with a metal-metal separation of 5.3 A. This structure is also inconsistent with a chemical mechanism for substrate hydrolysis that utilizes the bridging hydroxide as a base to abstract a proton from a water molecule loosely associated with the alpha-metal ion. Density functional theory (DFT) calculations support a reaction mechanism that utilizes the bridging hydroxide as the direct nucleophile in the hydrolysis of organophosphate esters by PTE.
ESTHER : Kim_2008_Biochemistry_47_9497
PubMedSearch : Kim_2008_Biochemistry_47_9497
PubMedID: 18702530

Title : Characterization of a phosphodiesterase capable of hydrolyzing EA 2192, the most toxic degradation product of the nerve agent VX - Ghanem_2007_Biochemistry_46_9032
Author(s) : Ghanem E , Li Y , Xu C , Raushel FM
Ref : Biochemistry , 46 :9032 , 2007
Abstract : Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a nonspecific diesterase that enables Escherichia coli to utilize alkyl phosphodiesters, such as diethyl phosphate, as the sole phosphorus source. The catalytic properties of GpdQ were determined, and the best substrate found was bis(p-nitrophenyl) phosphate with a kcat/Km value of 6.7 x 10(3) M-1 s-1. In addition, the E. aerogenes diesterase was tested as a catalyst for the hydrolysis of a series of phosphonate monoesters which are the hydrolysis products of the highly toxic organophosphonate nerve agents sarin, soman, GF, VX, and rVX. Among the phosphonate monoesters tested, the hydrolysis product of rVX, isobutyl methyl phosphonate, was the best substrate with a kcat/Km value of 33 M-1 s-1. The ability of GpdQ to hydrolyze the phosphonate monoesters provides an alternative selection strategy in the search of enhanced variants of the bacterial phosphotriesterase (PTE) for the hydrolysis of organophosphonate nerve agents. This investigation demonstrated that the previously reported activity of GpdQ toward the hydrolysis of methyl demeton-S is due to the presence of a diester contaminant in the commercial material. Furthermore, it was shown that GpdQ is capable of hydrolyzing a close analogue of EA 2192, the most toxic and persistent degradation product of the nerve agent VX.
ESTHER : Ghanem_2007_Biochemistry_46_9032
PubMedSearch : Ghanem_2007_Biochemistry_46_9032
PubMedID: 17630782

Title : Activation of the binuclear metal center through formation of phosphotriesterase-inhibitor complexes - Samples_2007_Biochemistry_46_3435
Author(s) : Samples CR , Raushel FM , DeRose VJ
Ref : Biochemistry , 46 :3435 , 2007
Abstract : Phosphotriesterase (PTE) from Pseudomonas diminuta is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphate nerve agents at rates approaching the diffusion-controlled limit. The proposed catalytic mechanism postulates the interaction of the substrate with the metal center and subsequent nucleophilic attack by the bridging hydroxide. X-band EPR spectroscopy was utilized to monitor the active site of Mn/Mn-substituted PTE upon addition of two inhibitors, diisopropyl methyl phosphonate and triethyl phosphate, and the product of hydrolysis, diethyl phosphate. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge were evaluated by measuring changes in the features of the EPR spectra. The EPR spectra support the proposal that the binding of substrate analogues to the binuclear metal center diminishes the population of hydroxide-bridged species. These results, in conjunction with previously published kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the beta-metal weakens the coordination of the hydroxide bridge to the beta-metal. The weakened coordination to the beta-metal ion increases the nucleophilic character of the hydroxide and is coupled to the increase in the electrophilic character of the substrate.
ESTHER : Samples_2007_Biochemistry_46_3435
PubMedSearch : Samples_2007_Biochemistry_46_3435
PubMedID: 17315951

Title : Sensitivity and specificity improvement of an ion sensitive field effect transistors-based biosensor for potato glycoalkaloids detection - Korpan_2006_J.Agric.Food.Chem_54_707
Author(s) : Korpan YI , Raushel FM , Nazarenko EA , Soldatkin AP , Jaffrezic-Renault N , Martelet C
Ref : Journal of Agricultural and Food Chemistry , 54 :707 , 2006
Abstract : Butyryl cholinesterase of different origin along with variations of the time of enzyme immobilization on the potentiometric transducer surface is offered to control the ion sensitive field effect transistor (ISFET)-based biosensor sensitivity. Because butyryl cholinesterase has been already used to develop the sensors for heavy metals, organophosphorus/carbamate pesticides, and steroidal glycoalkaloids analysis, the present study has been focused on the investigation and adjustment of the ISFET-based biosensor specificity exclusively to the glycoalkaloids. Utilization of ethylendiaminetetracetate (a complexon of heavy metal ions) and phosphotriesterase (a highly efficient catalyst for the hydrolysis of organophosphorus compounds) enabled the highly specific determination of glycoalkaloids at the background of lead and mercury (up to 500 microM of ions concentration) and paraoxon (up to 100 microM of pesticide concentration). The developed biosensor has been validated for glycoalkaloids detection in potato varieties cultivated in Ukraine, and the results obtained are compared to those measured by the methods of HPLC and TLC.
ESTHER : Korpan_2006_J.Agric.Food.Chem_54_707
PubMedSearch : Korpan_2006_J.Agric.Food.Chem_54_707
PubMedID: 16448172

Title : Detoxification of organophosphate nerve agents by bacterial phosphotriesterase - Ghanem_2005_Toxicol.Appl.Pharmacol_207_459
Author(s) : Ghanem E , Raushel FM
Ref : Toxicol Appl Pharmacol , 207 :459 , 2005
Abstract : Organophosphates have been widely used as insecticides and chemical warfare agents. The health risks associated with these agents have necessitated the need for better detoxification and bioremediation tools. Bacterial enzymes capable of hydrolyzing the lethal organophosphate nerve agents are of special interest. Phosphotriesterase (PTE) isolated from the soil bacteria Pseudomonas diminuta displays a significant rate enhancement and substrate promiscuity for the hydrolysis of organophosphate triesters. Directed evolution and rational redesign of the active site of PTE have led to the identification of new variants with enhanced catalytic efficiency and stereoselectivity toward the hydrolysis of organophosphate neurotoxins. PTE has been utilized to protect against organophosphate poisoning in vivo. Biotechnological applications of PTE for detection and decontamination of insecticides and chemical warfare agents are developing into useful tools. In this review, the catalytic properties and potential applications of this remarkable enzyme are discussed.
ESTHER : Ghanem_2005_Toxicol.Appl.Pharmacol_207_459
PubMedSearch : Ghanem_2005_Toxicol.Appl.Pharmacol_207_459
PubMedID: 15982683

Title : Access to the carbamate tunnel of carbamoyl phosphate synthetase - Kim_2004_Arch.Biochem.Biophys_425_33
Author(s) : Kim J , Raushel FM
Ref : Archives of Biochemistry & Biophysics , 425 :33 , 2004
Abstract : The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli revealed the existence of a molecular tunnel that has been proposed to facilitate the translocation of reaction intermediates between remotely located active sites. Five highly conserved glutamate residues, including Glu-25, Glu-383, Glu-577, Glu-604, and Glu-916, are close together in two clusters in the interior wall of the molecular tunnel that enables the intermediate carbamate to migrate from the site of synthesis to the site of utilization. Two arginines, Arg-306 and Arg-848, are located at either end of the carbamate tunnel and participate in the binding of ATP at each of the two active sites within the large subunit of CPS. The mutation of Glu-25 or Glu-577 results in a diminution in the overall rate of carbamoyl phosphate formation. Similar effects are observed upon mutation of Arg-306 and Arg-848 to alanine residues. The conserved glutamate and arginine residues may function in concert with one another to control entry of carbamate into the tunnel prior to phosphorylation to carbamoyl phosphate. The electrostatic environment of tunnel interior may help to stabilize the tunnel architecture and prevent decomposition of carbamate through protonation.
ESTHER : Kim_2004_Arch.Biochem.Biophys_425_33
PubMedSearch : Kim_2004_Arch.Biochem.Biophys_425_33
PubMedID: 15081891

Title : High resolution X-ray structures of different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta - Benning_2001_Biochemistry_40_2712
Author(s) : Benning MM , Shim H , Raushel FM , Holden HM
Ref : Biochemistry , 40 :2712 , 2001
Abstract : Phosphotriesterase, isolated from the soil-dwelling bacterium Pseudomonas diminuta, catalyzes the detoxification of organophosphate-based insecticides and chemical warfare agents. The enzyme has attracted significant research attention in light of its possible employment as a bioremediation tool. As naturally isolated, the enzyme is dimeric. Each subunit contains a binuclear zinc center that is situated at the C-terminal portion of a "TIM" barrel motif. The two zincs are separated by approximately 3.4 A and coordinated to the protein via the side chains of His 55, His 57, His 201, His 230, Asp 301, and a carboxylated Lys 169. Both Lys 169 and a water molecule (or hydroxide ion) serve to bridge the two zinc ions together. Interestingly, these metals can be replaced with cadmium or manganese ions without loss of enzymatic activity. Here we describe the three-dimensional structures of the Zn(2+)/Zn(2+)-, Zn(2+)/Cd(2+)-, Cd(2+)/Cd(2+)-, and Mn(2+)/Mn(2+)-substituted forms of phosphotriesterase determined and refined to a nominal resolution of 1.3 A. In each case, the more buried metal ion, referred to as the alpha-metal, is surrounded by ligands in a trigonal bipyramidal ligation sphere. For the more solvent-exposed or beta-metal ion, however, the observed coordination spheres are either octahedral (in the Cd(2+)/Cd(2+)-, Mn(2+)/Mn(2+)-, and the mixed Zn(2+)/Cd(2+)-species) or trigonal bipyramidal (in the Zn(2+)/Zn(2+)-protein). By measuring the anomalous X-ray data from crystals of the Zn(2+)/Cd(2+)-species, it has been possible to determine that the alpha-metal ion is zinc and the beta-site is occupied by cadmium.
ESTHER : Benning_2001_Biochemistry_40_2712
PubMedSearch : Benning_2001_Biochemistry_40_2712
PubMedID: 11258882

Title : Success of pyridostigmine, physostigmine, eptastigmine and phosphotriesterase treatments in acute sarin intoxication - Tuovinen_1999_Toxicology_134_169
Author(s) : Tuovinen K , Kaliste-Korhonen E , Raushel FM , Hanninen O
Ref : Toxicology , 134 :169 , 1999
Abstract : The acute toxicity of organophosphorus (OP) compounds in mammals is due to their irreversible inhibition of acetylcholinesterase (AChE) in the nervous system, which leads to increased synaptic acetylcholine levels. The protective actions of intravenously (i.v.) administered pyridostigmine, physostigmine, eptastigmine, and an organophosphate hydrolase, phosphotriesterase, in acute sarin intoxication were studied in mice. The acute intragastric (i.g.) toxicity (LD50) of sarin with and without the pretreatments was tested by the up-and-down method. The mice received pyridostigmine (0.06 mg/kg body weight), physostigmine (0.09 mg/kg body weight), the physostigmine derivative eptastigmine (0.90 mg/kg body weight) or phosphotriesterase (104 U/g, 10.7 microg/g body weight) 10 min prior to the i.g. administration of sarin. Physostigmine was also administered with phosphotriesterase. Phosphotriesterase was the most effective antidote in sarin intoxication. The LD50 value for sarin increased 3.4-fold in mice receiving phosphotriesterase. Physostigmine was the most effective carbamate in sarin exposure. The protective ratios of physostigmine and pyridostigmine were 1.5- and 1.2-1.3-fold, respectively. Eptastigmine did not give any protection against sarin toxicity. Both the phosphotriesterase and physostigmine treatments protected the brain AChE activities measured 24 h after sarin exposure. In phosphotriesterase and physostigmine-treated mice, a 4- and 2-fold higher sarin dose, respectively, was needed to cause a 50% inhibition of brain AChE activity. Moreover, the combination of phosphotriesterase-physostigmine increased the LD50 value for sarin 4.3-fold. The animals pretreated with phosphotriesterase-ephysostigmine tolerated four times the lethal dose in control animals, furthermore their survival time was 2-3 h in comparison to 20 min in controls. In conclusion, phosphotriesterase and physostigmine were the most effective treatments against sarin intoxication. However, eptastigmine did not provide any protection against sarin toxicity.
ESTHER : Tuovinen_1999_Toxicology_134_169
PubMedSearch : Tuovinen_1999_Toxicology_134_169
PubMedID: 10403635

Title : Stereochemical constraints on the substrate specificity of phosphotriesterase - Hong_1999_Biochemistry_38_1159
Author(s) : Hong SB , Raushel FM
Ref : Biochemistry , 38 :1159 , 1999
Abstract : A series of achiral, chiral, and racemic mixtures of paraoxon analogues containing various combinations of methyl, ethyl, isopropyl, or phenyl substituents were synthesized as probes of the stereochemical constraints within the active site of phosphotriesterase. The kinetic constants for these paraoxon analogues with the enzyme varied significantly with the size of substituents surrounding the phosphorus center. These results indicate that binding and catalysis depend significantly on the relative size and orientation of the two subsites that must accommodate the coordination of the alkyl or aryl substituents within the enzyme active site. Individual enantiomers of paraoxon analogues were also synthesized and the stereochemical specificity for phosphotriesterase determined. In general, the kinetic constants, kcat and kcat/Km, for the (-)-enantiomers of these phosphotriesters were 1-2 orders of magnitude greater than the (+)-enantiomers. In every case, the preferred isomer is of the SP-configuration. For example, the kcat/Km for SP-(-)-ethyl phenyl p-nitrophenyl phosphate is 1.8 x 10(8) M-1 s-1 but is only 1.8 x 10(6) M-1 s-1 for the RP-(+)-isomer. These results suggest that one enantiomer is positioned for hydrolysis more favorably than the other enantiomer. The inactivation of acetylcholinesterase with the same series of organophosphate nerve agents was also measured. The stereoisomer that more rapidly inactivates human acetylcholinesterase is hydrolyzed more slowly than its enantiomer by the phosphotriesterase.
ESTHER : Hong_1999_Biochemistry_38_1159
PubMedSearch : Hong_1999_Biochemistry_38_1159
PubMedID: 9930975

Title : Stereochemical preferences for chiral substrates by the bacterial phosphotriesterase - Hong_1999_Chem.Biol.Interact_119-120_225
Author(s) : Hong SB , Raushel FM
Ref : Chemico-Biological Interactions , 119-120 :225 , 1999
Abstract : The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate nerve agents such as paraoxon (diethyl p-nitrophenyl phosphate) with a turnover number of approximately 10(4) s(-1). The active site of the enzyme has been shown to be composed of a binuclear Zn2+ complex with a bridging hydroxide. The utilization of chiral phosphotriesters has demonstrated that the overall hydrolytic reaction occurs with net inversion of stereochemistry at the phosphorus center. The stereochemical constraints of the active site have been probed by the synthesis and characterization of paraoxon analogs. One or both of the two ethoxy substituents of paraoxon have been replaced with various combinations of methyl, isopropyl, or phenyl groups. Racemic mixtures and individual enantiomers were tested as substrates for the phosphotriesterase. In general, the kinetic constants (k(cat) and k(cat)/Km) for the (-)-enantiomers were one to two orders of magnitude greater than the (+)-enantiomer. Conversely, acetylcholinesterase was more rapidly inactivated by the (+)-enantiomers than the (-)-enantiomers. These results were examined in the context of the three-dimensional structure of the bacterial phosphotriesterase.
ESTHER : Hong_1999_Chem.Biol.Interact_119-120_225
PubMedSearch : Hong_1999_Chem.Biol.Interact_119-120_225
PubMedID: 10421456

Title : Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesterase - Watkins_1997_J.Biol.Chem_272_25596
Author(s) : Watkins LM , Mahoney HJ , McCulloch JK , Raushel FM
Ref : Journal of Biological Chemistry , 272 :25596 , 1997
Abstract : The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors. The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate. Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site. Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group. Eleven mutants were created using site-directed mutagenesis and purified to homogeneity. Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites. The single mutants W131K, F306K, and F306E were also constructed. Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate. Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant. There were significant increases in the Km for paraoxon for all mutants except F132H. Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold. Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K. Km values for acephate ranged from near wild type to a 5-fold increase. Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants. The Vmax value for the F132H/F306H mutant was increased to 3100 s-1. These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.
ESTHER : Watkins_1997_J.Biol.Chem_272_25596
PubMedSearch : Watkins_1997_J.Biol.Chem_272_25596
PubMedID: 9325279

Title : Inhibitors directed towards the binuclear metal center of phosphotriesterase - Hong_1997_J.Enzyme.Inhib_12_191
Author(s) : Hong SB , Raushel FM
Ref : J Enzyme Inhib , 12 :191 , 1997
Abstract : The potential roles in binding and catalysis for the binuclear metal center found within bacterial phosphotriesterase were evaluated by characterization of the inhibitory properties of 26 substrate and product mimetics. Phosphonates bearing monofluoro, difluoro, or hydroxyl substituents at the methylene position were found to be noncompetitive inhibitors with Ki values ranging from 0.6-9 mM versus the substrate paraoxon. Phosphoramidates did not significantly inhibit the enzyme. Diethyl phosphate and diethyl dithiophosphate inhibited the Cd-substituted enzyme with Ki values of 10 and 130 microM, respectively. The most effective inhibitor for either the cadmium or zinc substituted enzyme was found to be diethyl thiomethylphosphonate. The competitive inhibition constants for this compound were found to be 60 nM and 2.8 microM for the cadmium- and zinc-substituted enzyme, respectively. The tight binding is attributed to chelation of both metal ions simultaneously.
ESTHER : Hong_1997_J.Enzyme.Inhib_12_191
PubMedSearch : Hong_1997_J.Enzyme.Inhib_12_191
PubMedID: 9314115

Title : A combinatorial library for the binuclear metal center of bacterial phosphotriesterase - Watkins_1997_Proteins_29_553
Author(s) : Watkins LM , Kuo JM , Chen-Goodspeed M , Raushel FM
Ref : Proteins , 29 :553 , 1997
Abstract : Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had < 10(-5) of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.
ESTHER : Watkins_1997_Proteins_29_553
PubMedSearch : Watkins_1997_Proteins_29_553
PubMedID: 9408951

Title : Eptastigmine-phosphotriesterase combination in DFP intoxication - Tuovinen_1996_Toxicol.Appl.Pharmacol_140_364
Author(s) : Tuovinen K , Kaliste-Korhonen E , Raushel FM , Hanninen O
Ref : Toxicology & Applied Pharmacology , 140 :364 , 1996
Abstract : A novel therapy against organophosphate exposure, the combination of a carbamate eptastigmine and an organophosphate hydrolase (phosphotriesterase) was studied in mice against diisopropylfluorophosphate (DFP) (1.75 mg/kg) exposure. Mice received eptastigmine (0.9 mg/kg; iv) 10 min prior to the ip injection of DFP. Phosphotriesterase (83 U/g body weight) was injected iv 10 min after DFP. Eptastigmine (1.5 mg/kg; iv) inhibited the acetylcholinesterase activities in brain and erythrocytes for a longer time than physostigmine. Eptastigmine caused only minor changes in the behavior and activity of the animals, whereas physostigmine clearly reduced their activity for about 30 min. The eptastigmine pretreatment clearly supplemented the protective effect of phosphotriesterase against DFP: the plasma butyrylcholinesterase activity was doubled and the activity recovered faster than in animals treated with phosphotriesterase alone. In lung, butyrylcholinesterase activity was initially lower after eptastigmine-phosphotriesterase than phosphotriesterase treatment alone. However, the activity returned 24 hr later to normal in eptastigmine-phosphotriesterase-treated groups. With phosphotriesterase only, it recovered only to 75% of the control level. Presumably eptastigmine, by preventing the binding of DFP to cholinesterases, caused an elevation of free DFP levels in body fluids and promoted phosphotriesterase hydrolysis of DFP.
ESTHER : Tuovinen_1996_Toxicol.Appl.Pharmacol_140_364
PubMedSearch : Tuovinen_1996_Toxicol.Appl.Pharmacol_140_364
PubMedID: 8887453

Title : Phosphotriesterase, pralidoxime-2-chloride (2-PAM) and eptastigmine treatments and their combinations in DFP intoxication - Tuovinen_1996_Toxicol.Appl.Pharmacol_141_555
Author(s) : Tuovinen K , Kaliste-Korhonen E , Raushel FM , Hanninen O
Ref : Toxicology & Applied Pharmacology , 141 :555 , 1996
Abstract : The protective action of i.v. administered eptastigmine, an organophosphate hydrolase (phosphotriesterase), or pralidoxime-2-chloride (2-PAM) and their combination in acute diisopropylfluorophosphate (DFP) intoxication were evaluated in mice. The mice received the physostigmine derivative, eptastigmine (0.9 mg/kg body wt, i.v.), 10 min prior to the i.p. injection of DFP (1.8 mg/kg body wt). Phosphotriesterase (66 micromol/min x ml/g and 6 microg/g body wt) or 2-PAM (30 mg/kg body wt) were given i.v. 30 min after DFP. The animals also received atropine sc (37.5 mg/kg body wt) immediately after DFP. The cholinesterase (ChE) activities were not protected or reactivated by 2-PAM alone. The ChE activities in brain and plasma were protected by phosphotriesterase. Eptastigmine alone assisted the recovery of the brain ChE activities. Also the combination of eptastigmine-phosphotriesterase protected the brain enzymes. It did not, however, provide any additional protection compared with phosphotriesterase-treatment on its own. In brain, the combination of eptastigmine with 2-PAM resulted in partly restored enzyme activities 24 hr after DFP exposure. In plasma, eptastigmine did not prevent the inhibition of ChE by DFP. However, when it was combined with phosphotriesterase, it significantly promoted the recovery of plasma ChE activity. In lung and in erythrocytes, the various combinations of antidotes caused only minor changes in the ChE activities.
ESTHER : Tuovinen_1996_Toxicol.Appl.Pharmacol_141_555
PubMedSearch : Tuovinen_1996_Toxicol.Appl.Pharmacol_141_555
PubMedID: 8975781

Title : Phosphotriesterase--a promising candidate for use in detoxification of organophosphates - Tuovinen_1994_Fundam.Appl.Toxicol_23_578
Author(s) : Tuovinen K , Kaliste-Korhonen E , Raushel FM , Hanninen O
Ref : Fundamental & Applied Toxicology , 23 :578 , 1994
Abstract : The effect of phosphotriesterase (PTE) on cholinesterase (ChE) activities was studied with exposures to different organophosphates in mice. Paraoxon (PO) (1.0 mg/kg, ip) almost totally inhibited serum ChE activity. This activity, however, recovered to the normal level within 24 hr. The PTE pretreatment (16.8 U/animal, 2.5 micrograms/10 g body wt, iv 10 min before the organophosphate) accelerated this reactivation. The same phenomenon was also seen in vitro. In vitro with human serum, there was only minimal reactivation of the inhibited ChE. PTE, however, reactivated it significantly. The PTE-pretreated mice (168 U/animal, 30 micrograms/10 g body wt, iv) tolerated even 50 mg/kg of PO without showing any remarkable signs of intoxication. In PTE-untreated animals, however, PO doses as low as 1.0 and 1.5 mg/kg caused severe signs of poisoning. PTE (16.8 U/animal, 4 micrograms/10 g body wt, iv) reduced the inhibition of brain and serum ChE activities after PO and diisopropyl fluorophosphate exposure. In sarin and soman intoxications, PTE decreased only slightly the inhibition of ChE activities. The results indicate that PTE pretreatment given iv prevents the inhibition of ChE activities after certain organophosphates and it also hastens the recovery of activities after PO poisoning.
ESTHER : Tuovinen_1994_Fundam.Appl.Toxicol_23_578
PubMedSearch : Tuovinen_1994_Fundam.Appl.Toxicol_23_578
PubMedID: 7867909

Title : Phosphotriesterase decreases paraoxon toxicity in mice - Kaliste-Korhonen_1993_Toxicol.Appl.Pharmacol_121_275
Author(s) : Kaliste-Korhonen E , Ylitalo P , Hanninen O , Raushel FM
Ref : Toxicol Appl Pharmacol , 121 :275 , 1993
Abstract : The effect of phosphotriesterase (PTE) on the ip toxicity of paraoxon was studied in mice. The PTE preparation (0.1 ml; paraoxon-hydrolyzing activity, 1.5 mumol/min) was given iv. Cholinesterase activities were measured 2 hr after paraoxon administration. The PTE treatment, given 10 min before paraoxon, did not protect serum cholinesterase (ChE) against the inhibiting effect of paraoxon, but it clearly prevented the decrease of the brain ChE activity. In PTE-nontreated animals ChE was reduced by 60% at the paraoxon dose of 0.5 mg/kg, whereas in PTE-treated mice a significant reduction was not seen until a paraoxon dose of 2.0 mg/kg. The iv injection of PTE did prevent the decrease in brain ChE activity by paraoxon, when it was administered before or immediately after the paraoxon. PTE, injected 15 min after paraoxon, resulted in a minor protection in the brain ChE activities. The iv injection of PTE increased the serum paraoxon-hydrolyzing activity up to 5.1-fold. When the same amounts of PTE were administered ip, im, or sc, the increases in the hydrolyzing activities were 4.7-, 2.5-, and 1.8-fold, respectively. The activities returned to the normal level within 24 hr after the PTE. The elimination half-life of the activity of PTE administered iv was approximately 5.5 hr. In conclusion, PTE substantially prevents the toxicity of paraoxon in mice by hydrolyzing paraoxon in circulation.
ESTHER : Kaliste-Korhonen_1993_Toxicol.Appl.Pharmacol_121_275
PubMedSearch : Kaliste-Korhonen_1993_Toxicol.Appl.Pharmacol_121_275
PubMedID: 8394035

Title : Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta - Caldwell_1991_Biochemistry_30_7438
Author(s) : Caldwell SR , Newcomb JR , Schlecht KA , Raushel FM
Ref : Biochemistry , 30 :7438 , 1991
Abstract : The catalytic mechanism for the enzymatic hydrolysis of a series of paraoxon analogues by the phosphotriesterase from Pseudomonas diminuta has been determined. The Bronsted plots relating the pKa of the leaving group to the observed kinetic parameters, Vmax and V/Km, are both nonlinear. This observation is consistent with a change in the rate-limiting step from chemical to physical events as the pKa of the leaving group is decreased. This conclusion is confirmed by the effects of solvent viscosity on Vmax and V/Km for the same series of analogues. The data were fitted to the scheme E k1A in equilibrium k2 EA k3----EP k7----E'P k9----E + products where EA is the enzyme-substrate complex, EP is the enzyme-product complex, E'P is the enzyme-product complex after a viscosity-independent unimolecular reaction, and the values for k1, k2, k7, and k9 are 4.1 X 10(7) M-1 s-1, 2550 s-1, 3370 s-1, and 5940 s-1, respectively. The magnitude of the chemical step, represented by k3, is dependent on the pKa of the leaving group phenol as predicted by the Bronsted equation (log k3 = beta pKa + C) where beta = -1.8 and the constant (C) = 17.7. The magnitude of beta indicates that the transition state for substrate hydrolysis is very product-like.
ESTHER : Caldwell_1991_Biochemistry_30_7438
PubMedSearch : Caldwell_1991_Biochemistry_30_7438
PubMedID: 1649628

Title : Transition-state structures for enzymatic and alkaline phosphotriester hydrolysis - Caldwell_1991_Biochemistry_30_7444
Author(s) : Caldwell SR , Raushel FM , Weiss PM , Cleland WW
Ref : Biochemistry , 30 :7444 , 1991
Abstract : The primary and secondary 18O isotope effects for the alkaline (KOH) and enzymatic (phosphotriesterase) hydrolysis of two phosphotriesters, O,O-diethyl p-nitrophenyl phosphate (I) and O,O-diethyl O-(4-carbamoylphenyl) phosphate (II), are consistent with an associative mechanism with significant changes in bond order to both the phosphoryl and phenolic leaving group oxygens in the transition state. The synthesis of [15N, phosphoryl-18O]-, [15N, phenolic-18O]-, and [15N]-O,O-diethyl p-nitrophenyl phosphate and O,O-diethyl O-(4-carbamoylphenyl)phosphate is described. The primary and secondary 18O isotope effects for the alkaline hydrolysis of compound I are 1.0060 and 1.0063 +/- 0.0001, whereas for compound II they are 1.027 +/- 0.002 and 1.025 +/- 0.002, respectively. These isotope effects are consistent with the rate-limiting addition of hydroxide and provide evidence for a SN2-like transition state with the absence of a stable phosphorane intermediate. For the enzymatic hydrolysis of compound I, the primary and secondary 18O isotope effects are very small, 1.0020 and 1.0021 +/- 0.0004, respectively, and indicate that the chemical step in the enzymatic mechanism is not rate-limiting. The 18O isotope effects for the enzymatic hydrolysis of compound II are 1.036 +/- 0.001 and 1.0181 +/- 0.0007, respectively, and are comparable in magnitude to the isotope effects for alkaline hydrolysis, suggesting that the chemical step is rate-limiting. The relative magnitude of the primary 18O isotope effects for the alkaline and enzymatic hydrolysis of compound II reflect a transition state that is more progressed for the enzymatic reaction.
ESTHER : Caldwell_1991_Biochemistry_30_7444
PubMedSearch : Caldwell_1991_Biochemistry_30_7444
PubMedID: 1649629

Title : Purification and properties of the phosphotriesterase from Pseudomonas diminuta - Dumas_1989_J.Biol.Chem_264_19659
Author(s) : Dumas DP , Caldwell SR , Wild JR , Raushel FM
Ref : Journal of Biological Chemistry , 264 :19659 , 1989
Abstract : The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.
ESTHER : Dumas_1989_J.Biol.Chem_264_19659
PubMedSearch : Dumas_1989_J.Biol.Chem_264_19659
PubMedID: 2555328