Kinetic and chemical mechanism of α-isopropylmalate synthase from Mycobacterium tuberculosis
Kinetic and chemical mechanism of α-isopropylmalate synthase from Mycobacterium tuberculosis
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DOI：
10.1021/bi0606602
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发表时间：
2006-07-25
期刊：
影响因子：
2.9
通讯作者：
Blanchard, John S.
中科院分区：
文献类型：
--
作者：
de Carvalho, Luiz Pedro S.;Blanchard, John S.
结核分枝杆菌α-异丙基苹果酸合成酶（MtIPMS）催化乙酰辅酶A（AcCoA）与α-酮异戊酸（alpha-KIV）的缩合以及随后α-异丙基苹果酰-CoA的水解，生成产物CoA和α-异丙基苹果酸（alpha-IPM）。这是L-亮氨酸生物合成的第一个关键步骤。我们纯化了重组MtIPMS，并使用稳态动力学，同位素效应，同位素标记和H-1-NMR光谱的组合进行表征。该酶的α-酮酸特异性较窄，而酰基辅酶A特异性对乙酰辅酶A是绝对的。在不存在α-KIV的情况下，MtIPMS不烯醇化AcCoA的R质子，但缓慢水解酰基-CoA类似物。初始速度的研究，产品抑制，和死端抑制研究表明，MtIPMS遵循非快速平衡随机双双动力学机制，与首选的途径，三元复合物。MtIPMS需要两个催化碱基以获得最大活性（两者的pK（a）值均为约1.5）。6.7），我们认为一个催化AcCoA的去质子化和烯醇化，另一个活化参与α-异丙基苹果酰-CoA水解的水分子。初级氘和溶剂动力学同位素效应表明，有一个步骤后，化学是限速的，虽然，与穷人的底物，如丙酮酸，水解成为部分限速。我们的数据是不一致的建议，金属结合水参与水解。最后，我们的结果表明，α-异丙基苹果酰辅酶A的水解是直接的，没有形成环酐中间体。在此基础上，提出了MtIPMS催化反应的化学机理。
Mycobacterium tuberculosis alpha-isopropylmalate synthase (MtIPMS) catalyzes the condensation of acetyl-coenzyme A (AcCoA) with alpha-ketoisovalerate (alpha-KIV) and the subsequent hydrolysis of alpha-isopropylmalyl-CoA to generate the products CoA and alpha-isopropylmalate (alpha-IPM). This is the first committed step in L-leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and H-1-NMR spectroscopy. The alpha-keto acid specificity of the enzyme is narrow, and the acyl-CoA specificity is absolute for AcCoA. In the absence of alpha-KIV, MtIPMS does not enolize the R protons of AcCoA but slowly hydrolyzes acyl-CoA analogues. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random bi-bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pK(a) values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of alpha-isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate-limiting, although, with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of alpha-isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. On the basis of these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed.