Mechanism-based inactivation of phosphotriesterase by reaction of a critical histidine with a ketene intermediate.

Five alkynyl phosphate esters have been synthesized as probes of the active site structure of phosphotriesterase. These compounds have the potential to be converted by the enzyme to a highly reactive ketene intermediate which can then react with an active site nucleophile causing irreversible inhibition of the enzyme by formation of an inactive covalent adduct. All five compounds completely inactivate enzyme function in less than 15 s at pH 7.0. The partition rations of 1-hexynyl diethyl phosphate (I), 1-propynyl diethyl phosphate (II), 1-hexynyl diphenyl phosphate (III), 1-hexynyl dimethyl phosphate (IV), and ethynyl diethyl phosphate (V) fall in the range between 480 and 1700; thus, all five alkynyl phosphate esters work equally well as inactivators despite the differences in their structures. The rate constants for enzyme inactivation, kinact, are 1.7 s-1 with I, 1.3 s-1 with II, and 0.12 s-1 with IV. They compare well with the kcat for the Co-substituted phosphotriesterase; hence these compounds are good substrates. The stoichiometry of inhibitor bound to protein is 1:1, as determined by inactivation of the enzyme using the radiolabeled compound [3-14C]-1-propynyl diethyl phosphate. Addition of an exogenous nucleophile, azide, did not protect phosphotriesterase from being inactivated by the alkynyl phosphate esters, suggesting that the reactive intermediate produced from the inhibitor is not released from the enzyme surface prior to covalent labeling of the protein. Chemical and spectroscopic evidence suggests that a histidine residue is modified in the inactivation reaction. The inactivated phosphotriesterase can be reactivated by increasing the pH of the protein solution. N-Acylimidazoles are known to be easily hydrolyzed at alkaline pH values.(ABSTRACT TRUNCATED AT 250 WORDS)