Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl Transferases
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- 作者：Fuzhong Zhang and Paul J. Berti
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：May 16, 2006
- 年：2006
- 卷：45
- 期：19
- 页码：6027 - 6037
- 全文大小：179K
- 年卷期：v.45,no.19(May 16, 2006)
- ISSN：1520-4995
文摘
The role in catalysis of phosphate with AroA (enolpyruvyl shikimate 3-phosphate synthase)and MurA (enolpyruvyl UDP-GlcNAc synthase) was probed using phosphate analogues and an AroAmutant. Phosphate, the second reaction product, increases the reactivity of the enolpyruvyl products (EP-OR's) ~105-fold in the reverse reaction, forming phosphoenolpyruvate and R-OH (shikimate 3-phosphateor UDP-GlcNAc). Phosphate is intrinsically unreactive with EP-OR, raising the question of how AroAand MurA promote EP-OR reactivity. Eleven phosphate analogues were examined. All those with tetrahedralgeometries bound with AroA, except sulfate, while no nontetrahedral analogues did. Arsenate, vanadate,and fluorophosphate caused reactions of AroA and MurA with EP-OR's, yielding pyruvate and R-OH.Their kcat/KM values relative to phosphate were similar for both enzymes, ca. 100-fold worse for arsensate,200-fold worse for vanadate, and 5000-fold worse for fluorophosphate, implying similar interactions withboth enzymes. Examination of the arsenate-promoted reactions using [3'-3H]EP-OR's, 2H2O, and H218Oprovided evidence of an arseno-tetrahedral intermediate, analogous to the natural tetrahedral intermediate,proceeding to arsenoenolpyruvate, which spontaneously broke down to pyruvate and arsenate. The onlyphysicochemical property that appeared to be essential for reactivity of the analogues was the presenceof a proton. Titration of the intrinsic tryptophan fluorescence of the weakly active AroA mutant, Asp313Ala(D313A), demonstrated a fluorescence decrease upon enolpyruvyl shikimate 3-phosphate (EPSP) binding,and a further decrease upon binding of phosphate or arsenate to AroA_D313A·EPSP, suggesting a furtherconformational change. We are hopeful that understanding enzyme-phosphate interactions will make itpossible to design inhibitors that can use the high endogenous phosphate concentration in bacteria toenhance inhibitor binding.