Mechanism-based inactivation of phosphotriesterase by alkynyl phosphate esters and studies of the amidotransferase activity of E. coli carbamoyl phosphate synthetase

Abstract

Alkynyl phosphate esters were used to probe 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. The partition ratio of ethynyl diethyl phosphate was determined to be 1200. The stoichiometry of inhibitor bound to protein is 1: 1, as determined by inactivation of the enzyme using [ ] propynyl diethyl phosphate. Chemical and spectroscopic evidence suggests that a histidine residue is modified in the inactivation reaction. In order to identify which of the seven histidine residues in the native enzyme are required for inactivation, the kinetic properties of mutants in which each of the histidines were changed individually to asparagine were examined with 1-hexynyl diethyl phosphate. The H254N mutant could not be inactivated; no more than 60% of the initial activity was lost. These results suggest that His-254 is essential for the inactivation of phosphotriesterase and is likely to be the primary target in the wild-type enzyme for modification by 1-hexynyl diethyl phosphate. The results of an investigation regarding the mechanism of the amidotransferase reaction catalyzed by E. coli CPS are reported. The mechanism of the glutaminase activity is postulated to go via nucleophilic attack of the amide carbonyl of glutamine by an active site cysteine residue forining an acyl-enzyme intermediate which is hydrolyzed to form the products, ammonia and glutamate. The intermediate can be trapped and is acid stable. The steady state amount of intermediate was found to be 0.3 mol / mol CPS. A simple kinetic mechanism can be derived illustrating the steps involved in the glutamine hydrolysis reaction. The individual rate constants were determined by measuring the concentration dependence of the rate of formation and the rate of decay of the thioester intermediate. His-353 is essential for the formation of the intermediate, but not essential for substrate binding. Lastly, the CPS small subunit was isolated from a construction which expressed the small subunit and the N-terminal portion of the large subunit.