Engel Stanislav, Vyazmensky Maria, Vinogradov Michael, Berkovich Dvora, Bar-Ilan Ahuva, Qimron Udi, Rosiansky Yogev, Barak Ze'ev, Chipman David M
Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, 84105 Beer-Sheva, Israel.
J Biol Chem. 2004 Jun 4;279(23):24803-12. doi: 10.1074/jbc.M401667200. Epub 2004 Mar 25.
The thiamin diphosphate (ThDP)-dependent bio-synthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP(-)), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP(-) is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg(276)) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound HEThDP(-) with benzaldehyde to form phenylacetylcarbinol (Engel, S., Vyazmensky, M., Geresh, S., Barak, Z., and Chipman, D. M. (2003) Biotechnol. Bioeng. 84, 833-840) provides a new tool for studying the fate of HEThDP(-) in AHAS, since the formation of the new product has a very different dependence on active site modifications than does acetohydroxyacid acid formation. The effects of mutagenesis of four different residues in the site on the rates and specificities of the normal and unnatural reactions support a critical role for Arg(276) in the stabilization of the transition states for ligation of the incoming second ketoacid with HEThDP(-) and/or for the breaking of the product-ThDP bond. This information makes it possible to engineer the active site so that it efficiently and preferentially catalyzes a new reaction.
硫胺素二磷酸（ThDP）依赖性生物合成酶乙酰羟酸合酶（AHAS）催化丙酮酸脱羧，并使生成的与ThDP结合的二碳中间体羟乙基 - ThDP阴离子/烯胺（HEThDP(-)）与第二种酮酸发生特定缩合反应，形成乙酰乳酸或乙酰羟丁酸。虽然丙酮酸生成HEThDP(-)的机制已得到充分理解，但该酶在控制HEThDP(-)的碳连接反应中的作用尚不清楚。最近来自达格利比实验室的酵母AHAS晶体结构表明，一个精氨酸残基可能与第二种酮酸底物相互作用。对大肠杆菌AHAS同工酶II中这个完全保守的残基（Arg(276)）进行诱变，证实它是第二种酮酸快速和特异性反应所必需的。在突变蛋白中，反应通常快速的第二阶段成为限速步骤。结合的HEThDP(-)与苯甲醛发生竞争性替代非天然但立体特异性反应以形成苯乙酰甲醇（恩格尔，S.，维亚兹缅斯基，M.，格雷什，S.，巴拉克，Z.，以及奇普曼，D. M.（2003年）《生物技术与生物工程》84卷，833 - 840页），为研究AHAS中HEThDP(-)的命运提供了一种新工具，因为新产物的形成对活性位点修饰的依赖性与乙酰羟酸形成有很大不同。对该位点四个不同残基进行诱变对正常和非天然反应的速率及特异性的影响，支持了Arg(276)在稳定进入的第二种酮酸与HEThDP(-)连接的过渡态和/或产物 - ThDP键断裂方面的关键作用。这些信息使得对活性位点进行改造成为可能，从而使其能够高效且优先地催化新反应。
