MECHANISM-BASED INHIBITORS FOR THE INACTIVATION OF THE BACTERIAL PHOSPHOTRIESTERASE

1-Bromovinyl (I), Z-2-bromovinyl (II), 1,2-dibromoethyl (III), and a s eries of 4-(halomethyl)-2-nitrophenyl (IVa-c) diethyl phosphate esters were examined as substrates and mechanism-based inhibitors for the ba cterial phosphotriesterase. All of these compounds were found to act a s substrates for the enzyme, Inhibitor I rapidly inactivated the enzym e within 1 min, giving a partition ratio of 230, The newly formed cova lent adduct with inhibitor I was susceptible to hydrolysis at elevated values of pH and dissociation by NH2OH, Azide was not able to protect the enzyme from inactivation with inhibitor I, implying that the reac tive species was not released into solution prior to the inactivation event. The reactive species was proposed to be either an acyl bromide or a ketene intermediate formed by the enzymatic hydrolysis of inhibit or I. Compounds II and III were shown to be relatively poor substrates of phosphotriesterase and they did not induce any significant inactiv ation of the enzyme, The inhibitor, 4-(bromomethyl)-2-nitrophenyl diet hyl phosphate (IVa), was found to irreversibly inactivate the enzyme w ith a K-1 = 7.9 mM and k(inact) = 1.2 min(-1) at pH9.0. There was no e ffect on the rate of inactivation upon the addition of the exogenous n ucleophiles, azide, and NH2OH. The species responsible for the covalen t modification of the enzyme by IVa was most likely a quinone methide formed by the elimination of bromide from the phenolic intermediate. N MR experiments demonstrated that the quinone methide did not accumulat e in solution. The chloro (IVb) and fluoro (IVc) analogues did not ina ctivate the enzyme, These results suggest that the elimination of the halide ion from the phenolic intermediate largely determines the parti tion ratio for inactivation.