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Mechanistic, Inhibitory, and Mutagenic Studies of Inositol Dehydrogenase from Bacillus subtilis.

机译：枯草芽孢杆菌肌醇脱氢酶的机理，抑制和诱变研究。

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摘要

Inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis catalyzes the reversible NAD+-dependent oxidation of the axial hydroxyl group of myo-inositol to form 2-keto- myo-inositol, NADH and H+. IDH is the first enzyme in catabolism of myo-inositol, and Bacillus subtilis is able to grow on myo-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of myo-inositol.;In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the apo-IDH, holo-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD+-dependent to 6-fold preference for NADP + over NAD+ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed myo-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.
机译：枯草芽孢杆菌的肌醇脱氢酶（IDH，EC 1.1.1.18）催化肌醇的轴向羟基的NAD +依赖性可逆氧化，形成2-酮基肌醇，NADH和H +。 IDH是肌醇分解代谢中的第一种酶，枯草芽孢杆菌能够以肌醇作为唯一碳源生长。我们的实验室以前已经表明，该酶具有异常的活性位点，可以在肌醇的1L-4位上容纳较大的疏水取代基。在本论文中，对IDH的进一步表征进行了描述，重点是机理，抑制，动力学，底物结合和底物特异性的改变。动力学同位素效应研究表明，反应的化学步骤不受速率的限制。为了探测肌醇结合位点，合成了五个肌醇类似物并评价为竞争性抑制剂。最近，已经解决了脱辅基-IDH，全IDH和三元络合物的晶体结构。使用结构信息以及建模和序列比对方法，我们预测了酶的活性位点结构。基于这些预测，通过定点诱变，辅酶特异性从完全NAD +依赖性转变为NADP +的6倍于NAD +的6倍。因此，鉴定了用于辅酶识别的关键残基。除了辅酶特异性的改变，拟议的肌醇活性位点及其周围的11个氨基酸残基也被修饰以测试其作用，以增进我们对底物结合和激活的理解。

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  • 作者

    Zheng, Hongyan.;

  • 作者单位

    The University of Saskatchewan (Canada).;

  • 授予单位 The University of Saskatchewan (Canada).;
  • 学科 Chemistry Biochemistry.;Chemistry Organic.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 194 p.
  • 总页数 194
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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