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首页> 外文期刊>The FEBS journal >Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis
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Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis

机译：苹果酸异丙酯脱氢酶催化的结构和能量基础

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摘要

The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermusthermophilus in complex with Mn2+, its substrate isopropylmalate and its co-factor product NADH at 2.0 angstrom resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co-factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co-factor of approximately 3.0 angstrom. The structure further shows binding of a K+ ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high-level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3-isopropylmalate to the C4N atom of the pyridine ring of NAD(+) is easily possible, with an activation energy of approximately 15 kcal center dot mol(-1). The activation energy increases by approximately 4-6 kcal center dot mol(-1) when the K+ ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the epsilon-amino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3-isopropylmalate prior to hydride transfer by employing a low-barrier proton shuttle mechanism involving a water molecule.
机译：嗜热嗜热菌细菌3-异丙基苹果酸脱氢酶与Mn2 +，其底物异丙基苹果酸及其辅因子产物NADH的三维结构在2.0埃分辨率下具有完全封闭的构象。在两个结构域闭合时，使底物和辅因子精确地相对定向和紧密接近，底物的C 2原子与辅因子的吡啶环的C 4 N原子之间的距离约为3.0埃。该结构还显示了靠近活性位点的K +离子的结合，并为其已知的激活作用提供了解释。因此，这种结构对于酶活性复合物是极好的模拟。使用高级QM / MM计算，可以证明，在观察到的反应物排列中，很容易将氢化物从3-异丙基苹果酸的C2原子转移到NAD（+）吡啶环的C4N原子上。可能具有大约15 kcal中心点mol（-1）的活化能。当计算中省略K +离子时，活化能增加约4-6 kcal中心点mol（-1）。在最可能的情况下，在氢化物转移之前，Lys185的ε-氨基作为反应中的一般碱，通过使用涉及水的低势垒质子穿梭机制，在氢化物转移之前帮助3-异丙基苹果酸的去质子化反应分子。

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