BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 241, 294–296 (1997)ARTICLE NO. RC977569

Enzymatic Hydrolysis of Russian-VXby Organophosphorus Hydrolase

Vipin K. Rastogi,*,1 Joseph J. DeFrank,† Tu-chen Cheng,† and James R. Wild‡*GEO-Centers, Inc./ERDEC, Bldg. #E-3220, Aberdeen Proving Ground, Edgewood Area, Maryland 21010;†US Army ERDEC, Environmental Tech. Team, Aberdeen Proving Ground, Maryland 21010; and‡Biochemistry Department, Texas A&M University, College Station, Texas 77843

Received September 5, 1997

kcatÅ 0.3s01), this is the only characterized VX detoxi-The Russian-VX (R-VX) is the principle V-type nerve fying enzyme yet identified (Scheme 1).

agent in the chemical warfare (CW) arsenal of the For- A recent comprehensive study on non-enzymatic hy-mer Soviet Union. We here report the enzymatic hy- drolysis chemistry of V-type agents established thatdrolysis of the P-S bond of Russian-VX by organophos- both VX and R-VX appear to follow similar course ofphorus hydrolase (OPH) from Pseudomonas diminuta. degradation pattern following stoichiometric additionWhile the Michaelis constant, Km for R-VX (474 mM),

of water (6). The process is slow (rate constant ofwas similar to that for VX (434 mM), the Vmax for R-VX3.2111006s01 at 237C), and depending on the reaction(2.1 mmoles/mg/min) was about four-fold higher com-conditions can result in heterogeneous products, somepared to that for VX (0.56 mmoles/mg/min). A 50% inhi-of which are quite toxic (6). In contrast, the enzymaticbition in the rate of the enzymatic hydrolysis of R-VXhydrolysis of R-VX reported here is catalytic and re-was observed in the presence of 0.5% ethanol, isoamyl-sults in non-toxic products, 2-aminoethanethiol andalcohol, or isopropanol. The presence of acetonitrile,methylphosphonic acid.diethylene glycol, or methanol had marginal effects.

These results comprise the first demonstration of enzy-matic detoxification of R-VX. q 1997 Academic Press MATERIALS AND METHODS

Key Words: Russian-VX; enzymatic cleavage of P-Sbond; OPH; effect of organic solvents. OPH and VX hydrolysis assays. Purified cobalt-OPH (7) from

Pseudomonas diminuta was used in the present study. The R-VXhydrolysis assays were based on the detection of P-S bond cleavageproduct, 2-aminoethanethiol, utilizing the Ellman’s reagent (8) withfollowing modifications. Each assay included 10 mM TAPS buffer(pH 8.0), 10-20 mg OPH enzyme, 0.2 mM R-VX, 1.0 mM 5,5*-dithio-The V-type chemical warfare (CW) agents, VX [O- bis [2-nitrobenzoic acid] (DTNB), in a final volume of one ml. The

ethyl S-(2-diisopropylaminoethyl) methylphospho- rate of substrate hydrolysis was monitored using a Hewlett-Packardnothioate] and its isomer, Russian-VX, R-VX [O-iso- Model 8452 Diode Array spectrophotometer. The reactions were con-

ducted at 267C for up to one hr using the multicell kinetics modebutyl S-(2-diethylamino) methylphosphonothioate]with absorbance monitored at 412 nm. The molar extinction coeffi-are highly toxic, persistent, and included among thecient value of 14153 M01 was used for the thiol anion and enzymerespective stockpiles in the United States and the activity is presented as mmoles VX hydrolyzed/min/mg protein.

former Soviet Union (1-3). Enzymes catalyzing thehydrolysis of the P-S bond and thus resulting in de-

RESULTS AND DISCUSSIONtoxification of highly toxic V-type CW agents are ofinterest from the standpoint of developing an envi- Kinetic parameters. A number of buffers at differ-ronmentally-safe aqueous-based decontamination ent pH were first tested to determine the most effectivesystem. Recently, an organophosphorus hydrolase assay buffer. The highest specific activity was observed(OPH) enzyme from Pseudomonas diminuta was with TAPS buffer at pH 8 (results not shown). A time-demonstrated to hydrolyze the P-S bond of VX (4-5). course of R-VX hydrolysis in TAPS buffer is shown inAlthough OPH has a low catalytic turnover rate (4; Figure 1. In the presence of 20 mg OPH, about 120

mg R-VX was hydrolyzed within 2 hr. The R-VX/VXsubstrate was not hydrolyzed in the absence of OPH.1 To whom correspondence should be addressed. Fax: 410-612-

8661. E-mail: vkrastog@cbdcom-emh1.apgea.army.mil. Enzymatic assays with R-VX ranging between 0.166

0006-291X/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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SCHEME 1

and 5 mM concentrations were performed, and a Line- with diethyleneglycol, dimethylformamide and acetoni-trile resulting in similar inhibition. It was intriguingweaver-Burk plot was used to determine the kinetic

parameters. The Km and Vmax values for R-VX were 474 that the presence of small amounts of these commonsolvents had such marked effects on enzymatic hydro-mM and 2.1 mmoles/ml/min., respectively. Whereas, the

Km value for R-VX is similar to that reported for VX lysis of VX/R-VX. Finally, this inhibition appears to bea generalized effect on the active site of both OPH (P.(434 mM4), the maximum reaction velocity is about 4-

fold higher with R-VX compared to that for VX (2.1 vs. diminuta) and OPAA (Alteromonas JD6.5), since DFPhydrolysis was also subjected to a similar pattern of0.56 mmoles/ml/min). The turnover rate (kcat of 0.5s01)

for R-VX hydrolysis by OPH is low compared to the 465 inhibition (results not shown).and 3,170s01 values for DFP or paraoxon (9).

Effect of 0.5% organic solvents. In general, the CW CONCLUSIONSagents are water insoluble, and the stock solutions areroutinely prepared in isopropanol and then diluted to Enzymatic hydrolysis of R-VX by OPH resulting indesired concentration in assay buffer for laboratory non-toxic products is reported here. OPH enzyme isexperiments (final concentration of solvent range quite remarkable in its ability to hydrolyze a widebetween 0.5 and 5%). Significant inhibition (50%) by range of organophosphorus containing pesticides, G-isopropanol observed in a preliminary experiment type and V-type CW agents (4-5, 9). It was intriguingprompted us to test a number of other organic solvents to see a significant inhibitory effect of few common or-at 0.5% concentration. As seen in results summarized ganic solvents at 0.5% concentration. It is recom-in Figure 2, about 40-50% higher specific activity was mended that water-insoluble CW agent substrates beobserved for hydrolysis of R-VX compared to VX in the solubilized in methanol. Finally, demonstration of cata-control without organic solvents. The presence of iso- lytic hydrolysis of both V-type nerve agents, R-VX andamylalcohol, isopropanol, or ethanol resulted in 40-50% VX by OPH, greatly encourages the continued searchinhibition. Methanol was least inhibitory (15-20%),

FIG. 1. Effect of 0.5% organic solvents on P-S hydrolysis of R-VX and VX by OPH. The hydrolytic product, thiol-VX/R-VX, wasmeasured with Ellman reagent (as described in Materials and Meth- FIG. 2. Kinetics of Russian-VX hydrolysis by OPH. Assays were

performed in duplicate as described in Materials and Methods. Aver-ods). Each assay was done in duplicate and average values with errorbars from two experiments are shown. age values with error bars from two experiments are shown.

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4. Kolakowski, J. K., DeFrank, J. J., Lai, K., and Wild, J. R. (1994)for additional enzymes with improved hydrolytic capa-in ERDEC Scientific Conference on Chemical and Biological De-bilities against this class of agents.fense Research, APG, MD.

5. Hoskin, F. C. G., Walker, J. E., Dettbarn, W.-D., and Wild, J. R.REFERENCES (1995) Biochem. Pharmocol. 49, 711–715.

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7. Lai, K., Dave, K. I., and Wild, J. R. (1994) Biochemistry 269,2. Maynard, R. L., and Beswick, F. W. (1992) in Clinical and Exper-16579–16584.imental Toxicology of Organophosphates and Carbamates (Bal-

lantyne, B., and Marrs, T. C., Eds.), Oxford Butterworth-Heine- 8. Ellman, G. C., Courtney, K. D., Valentino, A., and Featherstone,mann, NY. R. M. (1961) Arch. Biochem. Biophys. 7, 70–77.

9. Lai, K., Stolowich, N. J., and Wild, J. R. (1995) Arch. Biochem.3. Yang, Y-C., Baker, J. A., and Ward, J. R. (1992) Chem. Rev. 92,1729–1743. Biophys. 318, 59–64.

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