Phosphotriesterase Research Articles

Stereospecificity in the enzymatic hydrolysis of cyclosarin (GF)

Enzymatic catalysis is one means of accelerating the rate of hydrolysis of G-type organophosphorus nerve agents. Here, the stereospecificity of the catalysis of cyclosarin (GF, O-cyclohexyl methylphosphonofluoridate) hydrolysis by several enzymes was investigated. Stereospecificity was not evident at 3 mM GF but was evident at 0.5 mM GF. The differential effect was apparently due to fluoride-catalyzed racemization of the substrate. Alteromonas sp. JD6.5 organophosphorus acid anhydrolase (OPAA), Alteromonas haloplanktis OPAA and the wild-type phosphotriesterase (PTE) enzymes were all found to catalyze preferentially the hydrolysis of the (+)GF isomer, as determined by GC analysis of the remaining unreacted (−)GF isomer. Acetylcholinesterase inhibition experiments showed the purified (−)GF isomer to be approximately twice as toxic as the racemic mixture. One PTE mutant, H254G/H259W/L303T, was found to reverse the native PTE stereospecificity and preferentially catalyze the hydrolysis of the (−)GF isomer, as shown by its complementation of Alteromonas sp. JD6.5 OPAA and by GC analysis of the remaining (+)GF isomer. This procedure also permitted the individual preparation of either of the two GF isomers by enzymatic degradation followed by extraction of the remaining isomer.

Phospholipid‐Based Catalytic Nanocapsules

AbstractThe encapsulation and catalytic efficiency of organophosphate hydrolyzing enzymes in polymer‐stabilized nanocapsules is reported. Polymerized vesicles—derived from a headgroup‐polymerizable phospholipid, 1,2‐dipalmitoyl‐sn‐glycero‐3‐phospho‐N‐(2‐hydroxymethyl)‐3,5‐divinylbenzamide (DPPE‐DVBA)—containing enzymes were used as catalytic nanocapsules. Three enzymes, organophosphorus hydrolase (OPH), phosphotriesterase (PTE), and organophosphorus acid anhydrolase (OPAA), were encapsulated in vesicles by incubating them with freeze‐dried vesicles at 55 °C, followed by intermittent vortex‐mixing. Enzyme‐containing vesicles, collected after gel‐filtration, were stabilized by photopolymerization at 254 nm to yield crosslinked catalytic nanocapsules. The nanocapsules containing OPH and PTE showed specific activities of 0.36 and 1.74 μmol mg–1 min–1, respectively, against methyl parathion (MPT), and OPAA‐containing nanocapsules showed a specific activity of 57.1 μmol mg–1 min–1 against diisopropylfluorophosphate. Freeze‐dried, OPH‐ and PTE‐containing nanocapsules showed retentions of 83 % and 85 % specific activity, respectively, upon redispersion in buffer solution. Three‐week, room‐temperature storage of OPH‐containing nanocapsules showed a retention of 18 % enzyme activity. Hydrolysis of MPT in crosslinked DPPE‐DVBA/OPH vesicles showed that hydrophobic MPT permeated through the bilayer membrane of the freeze‐dried nanocapsules, releasing the hydrolysis product para‐nitrophenol, which permeated back to the exogenous dispersion medium leaving the enzymes free to react with freshly permeated MPT in the interior of the nanocapsules.

Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence.

Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T < or =35 degrees C, followed by an activation phase near 45-48 degrees C and then inactivation which was completed at 60 degrees C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different T (m) values of 60.5-67 degrees C, 58-64 degrees C and 53-64 degrees C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.

Mechanism for the Hydrolysis of Organophosphates by the Bacterial Phosphotriesterase

Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5-10 was critical for substrate turnover. The pK(a) values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the alpha site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the alpha-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kinetic constants for IV were greater than those for II. The inverse thio effect is consistent with the polarization of the phosphoryl oxygen/sulfur bond via a direct ligation to the metal center. The rate enhancement is greater when Cd(2+) occupies the beta-metal-ion position. A series of alanine and asparagine mutations were used to characterize the catalytic roles of Asp233, His254, and Asp301. Mutations to either Asp233 or His254 resulted in an enhanced rate of hydrolysis for the sluggish substrate, diethyl p-chlorophenyl phosphate, and a decrease in the kinetic constants for paraoxon (I). These results are consistent with the existence of a proton relay from Asp301 to His254 to Asp233 that is used to ferry protons away from the active site with substrates that do not require activation of the leaving group phenol. A mechanism for the hydrolysis of organophosphates by the bacterial PTE has been proposed.

Coordination number of zinc ions in the phosphotriesterase active site by molecular dynamics and quantum mechanics.

We have run several molecular dynamics (MD) simulations on zinc-containing phosphotriesterase (PTE) with two bound substrates, sarin and paraoxon, and with the substrate analog diethyl 4-methylbenzylphosphonate. A standard nonbonded model was employed to treat the zinc ions with the commonly used charge of +2. In all the trajectories, we observed a tightly bound water (TBW) molecule in the active site that was coordinated to the less buried zinc ion. The phosphoryl oxygen of the substrate/inhibitor was found to be coordinated to the same zinc ion so that, considering all ligands, the less buried zinc was hexa-coordinated. The hexa-coordination of this zinc ion was not seen in the deposited X-ray pdb files for PTE. Several additional MD simulations were then performed using different charges (+1, +1.5) on the zinc ions, along with ab initio and density functional theory (DFT) calculations, to evaluate the following possibilities: the crystal diffraction data were not correctly interpreted; the hexa-coordinated zinc ion in PTE is only present in solution and not in the crystal; and the hexa-coordinated zinc ion in PTE is an artifact of the force field used. A charge of +1.5 leads to a coordination number (CN) of 5 on both zinc ions, which is consistent with the results from ab initio and DFT calculations and with the latest high resolution X-ray crystal structure. The commonly used charge of +2 produces a CN of 6 on the less buried zinc. The CN on the more buried zinc ion is 5 when the substrate/inhibitor is present in the simulation, and increases to 6 when the substrate/inhibitor is removed prior to the simulation. The results of both of the MD and quantum mechanical calculations lead to the conclusion that the zinc ions in the PTE active site are both penta-coordinated, and that the MD simulations performed with the charge of +2 overestimate the CN of the zinc ions in the PTE active site. The overall protein structures in the simulations remain unaffected by the change in zinc charge from +2 to +1.5. The results also suggest that the charge +1.5 is the most appropriate for the molecular dynamics simulations on zinc-containing PTE when a nonbonded model is used and no global thermodynamic conclusion is sought. We also show that the standard nonbonded model is not able to properly treat the CN and energy at the same time. A preliminary, promising charge-transfer model is discussed with the use of the zinc charge of +1.5.

Theoretical Determination of Two Structural Forms of the Active Site in Cadmium-Containing Phosphotriesterases

Two structural forms of the active site in Cd2+-containing phosphotriesterases (PTE), i.e., Cd2+/Cd2+−PTE and Zn2+/Cd2+−PTE, were determined by performing ab initio and density functional theory calculations on active site models. The calculated results indicate that the second bridging ligand in the active site of the recently reported high-resolution X-ray crystal structures of Cd2+/Cd2+−PTE and Zn2+/Cd2+−PTE should be a hydroxide ion, rather than a water molecule. The protonated active site structures of the Cd2+-containing enzymes are all significantly different from that of Zn2+/Zn2+−PTE. In Cd2+/Cd2+−PTE and Zn2+/Cd2+−PTE, the second bridging ligand always simultaneously coordinates to the two metal ions, whether it is a hydroxide ion or a water molecule. The pKa values calculated for the second bridging ligand are in good agreement with the observed kinetic pKa values for both the Cd2+- and Zn2+-substituted enzymes. The calculated results strongly support the conclusion that the observed kinetic pK...

The wild type bacterial Co 2+/Co 2+-phosphotriesterase shows a middle-range thermostability

The phosphotriesterase (PTE) from Pseudomonas diminuta, a metalloenzyme that catalyses the hydrolysis of organophosphorus pesticides and nerve agents, has been described as a remarkably heat-stable protein [Grimsley et al., Biochemistry 36 (1997), 14366–14374]. Because substitution of the naturally occurring zinc ions by cobalt ions was found to enhance the enzyme catalytic activity, we investigated the thermal stability of the Co 2+/Co 2+-PTE. This study, carried out using capillary electrophoresis under optimised conditions in the pH range 9–10 compatible with optimal enzyme activity, provided evidence for irreversible denaturation according to the Lumry–Eyring model. A temperature-induced conformational transition ( T m≈58°C) and an early growing of aggregates were observed. Comparison of UV spectra with heat-induced inactivation data clearly demonstrated that the PTE state populated above T m was neither native nor active. Differential scanning calorimetry showed only an exothermic trace due to aggregation of the denatured protein at T=76°C. Accordingly, the temperature-induced denaturation process of the PTE could be described by a consecutive reaction model, including formation of an intermediate with enhanced activity at T≈45°C and an inactive unfolded state populated at T≈58°C, which leads to denatured aggregates. Thus, the wild type Co 2+/Co 2+-PTE displays a middle-range thermostability. Hence, for decontamination purposes under extreme Earth temperatures, wild type and engineered mutants of PTE substituted with other metal cations should be evaluated.

Stereoselective Detoxification of Chiral Sarin and Soman Analogues by Phosphotriesterase

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S P- and R P-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R P-enantiomer of the sarin analogue ( k cat=2600 s −1) was the preferred substrate for the wild-type PTE relative to the corresponding S P-enantiomer ( k cat=290 s −1). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k cat values for the R P- and S P-enantiomers were 410 and 4200 s −1, respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R P R C-stereoisomer of the soman analogue was the preferred substrate for PTE. The k cat values for the soman analogues were measured as follows: R P R C, 48 s −1; R P S C, 4.8 s −1; S P R C, 0.3 s −1, and S P S C, 0.04 s −1. With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k cat values for the soman analogues were found to be as follows: R P R C, 0.3 s −1; R P S C, 0.3 s −1; S P R C, 11 s −1, and S P S C, 2.1 s −1. Prior investigations have demonstrated that the S P-enantiomers of sarin and soman are significantly more toxic than the R P-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.

Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris

Background: Phosphotriesterases (PTE) are enzymes capable of detoxifying organophosphate-based chemical warfare agents by hydrolysis. One subclass of these enzymes comprises the family of diisopropylfluorophosphatases (DFPases). The DFPase reported here was originally isolated from squid head ganglion of Loligo vulgaris and can be characterized as squid-type DFPase. It is capable of hydrolyzing the organophosphates diisopropylfluorophosphate, soman, sarin, tabun, and cyclosarin. Results: Crystals were grown of both the native and the selenomethionine-labeled enzyme. The X-ray crystal structure of the DFPase from Loligo vulgaris has been solved by MAD phasing and refined to a crystallographic R value of 17.6% at a final resolution of 1.8 Å. Using site-directed mutagenesis, we have structurally and functionally characterized essential residues in the active site of the enzyme. Conclusions: The crystal structure of the DFPase from Loligo vulgaris is the first example of a structural characterization of a squid-type DFPase and the second crystal structure of a PTE determined to date. Therefore, it may serve as a structural model for squid-type DFPases in general. The overall structure of this protein represents a six-fold β propeller with two calcium ions bound in a central water-filled tunnel. The consensus motif found in the blades of this β propeller has not yet been observed in other β propeller structures. Based on the results obtained from mutants of active-site residues, a mechanistic model for the DFP hydrolysis has been developed.

Mobility of the active site bound paraoxon and sarin in zinc-phosphotriesterase by molecular dynamics simulation and quantum chemical calculation.

The kinetic data published on phosphotriesterase (PTE), with various complexed metals, clearly indicates that the P=O and P=S bonds of phosphotriester and thiophosphotriester substrates, respectively, are strongly polarized by one or both of the active site complexed metal ions. However, this observation is not consistent with the three-dimensional X-ray crystal structure of zinc-substituted PTE with active site bound substrate analogue diethyl 4-methylbenzylphosphonate. In this structure, the distance between the phosphoryl oxygen and the nearest zinc is 3.4 A, a distance too large to afford strong polarization. In the present paper, the geometry and mobility of various PTE active site-substrate complexes are examined by performing both molecular dynamics (MD) simulations and quantum mechanical calculations. Two known substrates are considered, paraoxon and sarin, although their turnover rates vary about 100-fold. The results indicate that PTE forms a complex with either substrate in which the phosphoryl oxygen becomes strongly coordinated with the less buried zinc atom. It is shown that the geometry of the active site is changed when the protein is immersed in a water bath and relaxed by MD. The most substantial conformational change is the opening of the gateway in a pocket where the location of the leaving group is expected. The opening is observed for the pure enzyme as well as for the enzyme/substrate complexes and it ranges from 11 to 18 A. It is also shown that the pockets, in which the substrate substituents are localized, exhibit different flexibility and interact with the substrate with coordinated conformational adjustments.

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues.

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase.

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.

Partial characterization of phosphotriesterase activity from the earthworm, Eisenia andrei

Phosphotriesterase (PTE) receives attention because it seems to be associated with the detoxification of organophosphorous pesticides and organophosphate resistance mechanism. In order to understand the biodegradation of phosphotriester pesticides and its significance in the earthworm, a major non-target animal of pesticides, selected properties of phosphotriesterase activity derived from the crude extract of Eisenia andrei were investigated. PTE activity appeared to be primarily localized in intestinal tissues. The highest level of PTE activity was found in epithelial tissue. The native molecular weight of earthworm PTE was 260 kDa and the isoelectric point was approximately 4. The optimal pH was approximately 9. The earthworm PTE had a substrate affinity for paraoxon with K m value in the millimolar range. The presence of EGTA and EDTA completely abolished the activity and replacement of Ca 2+ ion restored activity to greater than 95%, suggesting that Ca 2+ ion is essential to maintain the activity.

The Binding of Substrate Analogs to Phosphotriesterase

Phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the detoxification of organophosphates such as the widely utilized insecticide paraoxon and the chemical warfare agent sarin. The three-dimensional structure of the enzyme is known from high resolution x-ray crystallographic analyses. Each subunit of the homodimer folds into a so-called TIM barrel, with eight strands of parallel beta-sheet. The two zinc ions required for activity are positioned at the C-terminal portion of the beta-barrel. Here, we describe the three-dimensional structure of PTE complexed with the inhibitor diisopropyl methyl phosphonate, which serves as a mimic for sarin. Additionally, the structure of the enzyme complexed with triethyl phosphate is also presented. In the case of the PTE-diisopropyl methyl phosphonate complex, the phosphoryl oxygen of the inhibitor coordinates to the more solvent-exposed zinc ion (2.5 A), thereby lending support to the presumed catalytic mechanism involving metal coordination of the substrate. In the PTE-triethyl phosphate complex, the phosphoryl oxygen of the inhibitor is positioned at 3.4 A from the more solvent-exposed zinc ion. The two structures described in this report provide additional molecular understanding for the ability of this remarkable enzyme to hydrolyze such a wide range of organophosphorus substrates.

Self-assembly of the binuclear metal center of phosphotriesterase.

The active site of the bacterial phosphotriesterase (PTE) from Pseudomonas diminuta contains two divalent metal ions and a carboxylated lysine residue. The native enzyme contains two Zn(2+) ions, which can be replaced with Co(2+), Cd(2+), Ni(2+), or Mn(2+) without loss of catalytic activity. Carbon dioxide reacts with the side chain of lysine-169 to form a carbamate functional group within the active site, which then serves as a bridging ligand to the two metal ions. The activation of apo-PTE using variable concentrations of divalent metal ions and bicarbonate was measured in order to establish the mechanism by which the active site of PTE is self-assembled. The time courses for the activation of apo-PTE are pseudo-first-order, and the observed rate constants are directly proportional to the concentration of bicarbonate. In contrast, the apparent rate constants for the activation of apo-PTE decrease as the concentrations of the divalent cations are increased and then become constant at higher concentrations of the divalent metal ions. These results are consistent with a largely ordered kinetic mechanism for the assembly of the binuclear metal center where CO(2)/bicarbonate reacts with the apo-PTE prior to the binding of the two metal ions. When apo-PTE is titrated with 0-8 equiv of Co(2+), Cd(2+), or Zn(2+), the concentration of activated enzyme increases linearly until 2 equiv of metal ion is added and then remains constant at elevated levels of the divalent cations. These results are consistent with the synergistic binding of the two metal ions to the active site, and thus the second metal ion binds more tightly to the protein than does the first metal ion. Measurement of the mean dissociation constant indicates that metal binding to the binuclear metal center is strong [(K(alpha)K(beta))(1/2) = 6.0 x 10(-)(11) M and k(off) = 1.5 x 10(-)(3) min(-)(1) for Zn(2+)]. The removal of the carbamate bridge through the mutagenesis of Lys-169 demonstrates that the carbamate bridge is required for both efficient catalysis and overall stability of the metal center.

Improvement of enantioselectivity of chiral organophosphate insecticide hydrolysis by bacterial phosphotriesterase.

The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp. can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be an effective catalyst for the degradation of toxic organophosphate esters. Ethyl 4-nitrophenyl phenylphosphonothioate (EPN) is a chiral organophosphate. Optical isomers of EPN show differences in their toxicity. R-EPN is known to be more toxic to hens and houseflies than S-EPN. We determined the Ki value of each enantiomer toward electric eel acetylcholinesterase. R-EPN (Ki = 6 microM) inhibited acetylcholinesterase much more effectively than S-EPN (Ki = 52 microM) did in vitro. Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively. When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration. At 20% DMSO, the maximum activity was observed. The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed. The Km value for R-EPN decreased from 0.24 to 0.03 mM, and the Vmax value increased from 0.25 to 0.60 U/mg of protein. Vmax/Km values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.

Ab initio determination of the structure of the active site of a metalloenzyme: Metal substitution in phosphotriesterase using density functional methods

A number of previous studies have determined that a metalloenzyme active site is inherently determined by the ligands and metal and only weakly perturbed by the surrounding protein environment. This conclusion is now being tested for several families of bimetallic enzymes. Metal substitution is examined for phosphotriesterase (PTE) with all possible substitutions of zinc and cadmium in the two sites. A new density functional theory (DFT) functional is used in this study which has yielded accurate structures and thermochemistry for molecules made up of first and second row atoms. Comparisons between gradient-optimized Hartree–Fock and DFT structures are in good agreement, suggesting this functional is useful in studying transition metal enzyme active sites. Good agreement between the X-ray and in vacuo optimized structures is also obtained for the Zn–Zn and Cd–Cd enzymes. This supports the analysis of the PTE active site as an inherent complex and suggests that accurate structures can be obtained for other metal substitutions for which no experimental structures are available. A unique zinc/cadmium hybrid was observed experimentally, and the structure of this active site is theoretically predicted. The polarization of bound water at this very polar active site is very large, suggesting it is the reactive nucleophile. ©1999 John Wiley & Sons, Inc. Int J Quant Chem 75: 289–299, 1999

Enzymes hydrolyzing organophosphates as potential catalytic scavengers against organophosphate poisoning

Enzymes hydrolyzing organophosphates could be used as catalytic scavengers for treatment of organophosphate poisoning and for decontamination. Two organophosphorus hydrolases (OPH) were selected: the Flavobacterium sp./ Pseudomonas diminuta phosphotriesterase (PTE) and human paraoxonase (HuPON). Genes encoding these enzymes were cloned and functional recombinant enzymes expressed. PTE was expressed in E. coli. Natural HuPON was purified from human plasma; recombinant HuPON was expressed in human embryonic kidney 293 T cells. Although HuPON displays interesting catalytic properties, a site-directed mutagenesis program was undertaken to improve its catalytic efficiency. PTE has high efficiency in hydrolysis of organophosphates, including nerve agents. PTE injected in rat has a half-life of 100 min. However, to overcome pharmacokinetic problems of injected OPH and/or immunological incompatibility, the model enzyme (recombinant PTE) was immobilized onto a hollow-fiber reactor. This reactor designed for extracorporeal blood circulation is under experimentation for post-exposure detoxification.

Characterization of a phosphotriesterase from genetically‐engineered Escherichia coli

A phosphotriesterase (PTE) capable of hydrolyzing organophosphate esters was purified from Escherichia coli strain DH‐5α carrying a cloned opd gene from Flavobacterium. The effects of pH, temperature and metal ion concentrations on enzyme stability and activity were investigated. Optimum conditions for PTE's catalytic activity were determined to be 35°C and pH 8.5. Protein‐metal equilibrium binding experiments showed that PTE could accommodate two equivalents of Co2+ or Zn2+ ions. PTE protein was found to have higher affinity for Co2+. In addition, Co2+ was found to possess the most positive effects in maintaining and restoring PTE's stability and catalytic activity when compared to other divalent metal ions. Assessment of the feasibility of PTE operation in a practical environment was performed in a system designed to mimic a continuously stirred tank reactor (CSTR) with different solution compositions in the flow reservoir. PTE was deactivated in 24 hours when the inflow solution contained 5% ethanol or 1 mM EDTA, while it retained one third of its initial activity in a deionized water stream. When the inflow solution contained 1 mM Co2+, PTE was found to retain activity throughout the 24‐hour experiment.

A combinatorial library for the binuclear metal center of bacterial phosphotriesterase.

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.