Structural and kinetic determinants of aldehyde reduction by aldose reductase
Structural and kinetic determinants of aldehyde reduction by aldose reductase
复制标题
DOI：
10.1021/bi981794l
复制
发表时间：
1999-01-05
期刊：
影响因子：
2.9
通讯作者：
Bhatnagar, A
中科院分区：
文献类型：
--
作者：
Srivastava, S;Watowich, SJ;Bhatnagar, A
醛糖还原酶（AR）是醛酮还原酶超家族的成员。由于其在高血糖和高渗应激期间催化葡萄糖形成山梨糖醇的能力，AR的降醛特性已被认为是其主要的生理和病理功能。尽管如此，AR是葡萄糖还原的不良催化剂，并显示出碳水化合物结合蛋白质意想不到的活性位点性质。因此，我们研究了AR与一系列天然存在的醛的催化性质，其疏水性与酶的大非极性活性位点相容。我们的研究结果表明，重组人AR是一种有效的催化剂，用于减少中长链无支链的饱和和不饱和醛。该酶对饱和醛如己醛和不饱和醛如反式-2-辛烯醛、壬烯醛及其4-羟基衍生物具有选择性。短链醛如丙醛和丙烯醛的还原效率较低。然而，丙烯醛或其谷胱甘肽缀合物（GS-丙醛）的支链衍生物以高效率被还原。在不存在NADPH的情况下，α、β不饱和醛引起酶的共价修饰。基于AR的野生型和定点突变体的电喷雾质谱分析，其中溶剂暴露的半胱氨酸被丝氨酸单独取代，修饰位点被鉴定为活性位点残基Cys 298。然而，不饱和醛并不修饰与NADPH结合的酶，并且在催化过程中不使酶变性。模拟研究表明，AR的大疏水活性位点可以容纳大量的醛，而不改变结合位点的结构或侧链的移动，由于长烷基链或非极性取代基的高疏水性似乎稳定醛底物与酶的相互作用。显然，这种疏水相互作用提供了通过氢键可实现的量级的底物选择性和催化效率。由于AR减少的几种醛是环境和药理学污染物或脂质过氧化产物，本研究提供了未来研究AR在调节醛代谢中的作用的基础，特别是在与氧化应激和/或醛毒性相关的病理状态下。
Aldose reductase (AR) is a member of the aldo-keto reductase superfamily. Due to its ability to catalyze the formation of sorbitol from glucose during hyperglycemic and hypertonic stress, the aldose-reducing property of AR has been accepted as its main physiological and pathological function. Nonetheless, AR is a poor catalyst for glucose reduction and displays active-site properties unexpected of a carbohydrate-binding protein. We, therefore, examined the catalytic properties of AR with a series of naturally occurring aldehydes, compatible in their hydrophobicity to the large apolar active site of the enzyme. Our results show that recombinant human AR is an efficient catalyst for the reduction of medium- to long-chain unbranched saturated and unsaturated aldehydes. The enzyme displayed selective preference For saturated aldehydes, such as hexanal, and unsaturated aldehydes, such as trans-2-octenal and nonenal as well as their 4-hydroxy derivatives. Short-chain aldehydes such as propanal and acrolein were reduced less efficiently. Branched derivatives of acrolein or its glutathione conjugate (GS-propanal) were, however, reduced with high efficiency. In the absence of NADPH, the alpha, beta unsaturated aldehydes caused covalent modification of the enzyme. On the basis of electrospray mass spectrometric analysis of the wild-type and site-directed mutants of AR tin which the solvent exposed cysteines were individually replaced with serine), the site of modification was identified to be the active-site residue, Cys 298. The unsaturated aldehydes, however, did not modify the enzyme bound to NADPH and did not inactivate the enzyme during catalysis. Modeling studies indicate that the large hydrophobic active site of AR can accommodate a large number of aldehydes without changes in the structure of the binding site or movement of side chains, High hydrophobicity due to long alkyl chains or apolar substituents appears to stabilize the interaction of the aldehyde substrates with the enzyme. Apparently, such hydrophobic interactions provide substrate selectivity and catalytic efficiency of the order achievable by hydrogen bonding. Since several of the aldehydes reduced by AR are either environmental and pharmacological pollutants or products of lipid peroxidation, the present studies provide the basis of future investigations on the role of AR in regulating aldehyde metabolism particularly under pathological states associated with oxidative stress and/or aldehyde toxicity.