Stereoselective hydrolysis of organophosphate nerve agents by the bacterial phosphotriesterase

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.

Figure 1

Graphic representation of the binding pockets within the active site of PTE. The small pocket consists of Gly-60, Ile-106, Leu-303, and Ser-308. The large pocket consists of His-254, His-257, Leu-271, and Met-317. The leaving group pocket is surrounded by Trp-131, Phe-132, Phe-306, and-Tyr 309.

Figure 2

Time course for the hydrolysis of racemic 5 using two mutants of PTE. The total concentration of compound 5 was determined by the complete hydrolysis using 0.1 M KOH. The hydrolysis of the S_P-enantiomer of racemic 5 was initiated with 165 nM H254G/H257W/L303T of PTE. After 10 minutes, the G60A mutant of PTE was added to hydrolyze the R_P-enantiomer.

Figure 3

Hydrolysis of 250 μM racemic GB followed by ITC. All reactions were initiated by injection of 5 μL of GB into a volume of 200 μL and followed with a MicroCal iTC₂₀₀ isothermal calorimeter. (A) Hydrolysis of racemic GB by 30 nM wild-type PTE as a function of time. The data are fit to single exponential. (B) Hydrolysis of GB by 60 nM G60A as function of time. The data are fit to the sum of two exponentials. (C) Hydrolysis of racemic GB by 10 nM H257Y/L303T mutant as function of time. The data are fit to the sum of two exponentials.

Figure 4

Separation of stereoisomers of racemic GD by gas chromatography.

Figure 5

Hydrolysis of 300 μM GD. All reactions were conducted in a volume of 200 μL and initiated by the injection of 6 μL of GD into the enzyme solution. Hydrolysis of GD (panels A, C, E, and G) was followed by monitoring the heat liberated as a function of time. Following each experiment the unreacted fraction of GD was analyzed by gas chromatography with a chiral separation column (panels B, D, F, and H). Order of elution from the gas chromatography column is S_PR_C (13 min), R_PR_C (14.1 min), S_PS_C (14.5 min), R_PS_C (15 min). (A) Hydrolysis of GD by 800 nM wild-type PTE as a function of time. Line is fit to single exponential. (B) Analysis of GD remaining following reaction with wild-type PTE. (C) Hydrolysis of GD by 200 nM G60A as a function of time. Line is a fit to the sum of 2 exponentials. (D) Analysis of unreacted GD following reaction with G60A. (E) Hydrolysis of GD by 600 nM H254G/H257W/L303T as a function of time. The line represents a fit of the data to a single exponential. (F) Analysis of uneacted GD remaining following reaction with H254G/H257W/L303T. (G) Hydrolysis of GD by100 nM H257Y/L303T as function of time. The line is a fit to the sum of two exponentials. (H) Analysis of unreacted GD remaining following reaction with H257Y/L303T.

Figure 6

Bar graph illustrating changes in the value of k_cat/K_m for the S_P-enantiomers of compounds 2, 3, 4, and 5 catalyzed by the wild-type and mutant forms of PTE.

Scheme 1

Structures of chemical warfare agents.

Scheme 2

Hydrolysis of paraoxon by phosphotriesterase.

Scheme 3

Structures of organophosphate substrates.