Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl Transferases

Abstract

<p> The two carboxyvinyl transferases MurA and AroA are essential for bacterial survival, and are proven or potential antibiotic targets. The reactions they catalyze are chemically challenging, involving protonation of an ethylene group in the first step, and deprotonation of a methyl in the second step. In order to probe how the enzymes promote these reactions, the reverse reactions from enolpyruvyl compounds (EP-OR) plus phosphate to phosphoenolpyruvate (PEP) plus R-OH were investigated, and compared with EP-OR hydrolysis reactions catalyzed by phosphate analogues. </p> <p> Thirteen phosphate analogues were used to study EP-OR hydrolysis. Among these phosphate analogues, many could bind to the free enzymes, but only three could promote hydrolysis. The products were pyruvate and the corresponding alcohol (S3P in AroA/EPSP reaction and UDP-GicNAc in MurA/EP-UDP-GicNAc reaction). The most effective analogue was arsenate. The mechanism of the arsenate-promoted reaction was examined in detail. The hydrolysis reaction proceeded though an arseno-tetrahedral intermediate with AroA, a similar reaction pathway to the natural reaction. This arseno-tetrahedral intermediate was converted to arsenoenolpyruvate and hydrolyzed spontaneously. MurA also likely catalyzed arseno-tetrahedral intermediate formation, and appeared to catalyze arsenoenolpyruvate breakdown, though it is possible that it was a bystander in the reaction, with the tetrahedral intermediate being formed by water attack on C2 of EP-UDP-GicNAc. There was a fast solvent exchange step before EP OR was converted to arseno-THI by AroA or MurA. This strongly indicated an oxacarbenium ion like intermediate before the arseno-tetrahedral intermediate. </p> <p> The catalytic machinery for stabilizing such an unstable oxacarbenium ion like intermediate was investigated by studying ligand binding. Based on information from all the phosphate analogues, there were evidence that the enzyme undergoes an conformational change upon binding with phosphate, by which EPSP was distorted into an oxacarbenium ion like intermediate. </p>