Henehan G T, Chang S H, Oppenheimer N J
Department of Pharmaceutical Chemistry S-926, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1995 Sep 26;34(38):12294-301. doi: 10.1021/bi00038a025.
The ability of Drosophila alcohol dehydrogenase (D-ADH) to catalyze the oxidation of aldehydes to carboxylic acids has been re-examined. Prior studies are shown to have been compromised by a nonenzymic reaction between the aldehydic substrates and amine-containing buffers, e.g., glycine or Tris, and an amine-catalyzed addition of aldehyde to NAD+. These reactions interfere with spectrophotometric assays for monitoring aldehyde oxidation and obscure the nature and scope of D-ADH-catalyzed aldehyde oxidation, particularly at physiological pH. Use of nonreactive buffers, such as pyrophosphate or phosphate, and 1H NMR spectroscopy to monitor all the components of the reaction mixture reveals the facile dismutation of aldehydes into equimolar quantities of the corresponding acids and alcohols at both neutral and high pH. At high pH, dismutation is accompanied by a small burst of NADH production to a steady-state concentration ( < 10 microM) that represents a partitioning between NADH dissociation and aldehyde reduction. The increase in A340 is therefore not a direct measure of the aldehyde oxidation reaction, and the resulting kinetic values cannot be compared to those for alcohol dehydrogenation. The present results for D-ADH, combined with data from the literature, establish that aldehyde oxidation, manifest as dismutation, is a widespread property of alcohol dehydrogenases with potential physiological importance in alcohol metabolism and aldehyde detoxification.
果蝇乙醇脱氢酶（D-ADH）催化醛氧化为羧酸的能力已被重新研究。先前的研究表明，醛底物与含胺缓冲液（如甘氨酸或Tris）之间的非酶促反应以及胺催化的醛与NAD⁺的加成反应影响了这些研究。这些反应干扰了用于监测醛氧化的分光光度法测定，并掩盖了D-ADH催化的醛氧化的性质和范围，尤其是在生理pH值下。使用无反应性缓冲液（如焦磷酸盐或磷酸盐）以及¹H NMR光谱来监测反应混合物的所有成分，结果表明，在中性和高pH值下，醛都能轻松歧化为等摩尔量的相应酸和醇。在高pH值下，歧化伴随着少量NADH的短暂生成，达到稳态浓度（<10 μM），这代表了NADH解离与醛还原之间的分配。因此，A340的增加并非醛氧化反应的直接度量，所得的动力学值也无法与乙醇脱氢反应的动力学值进行比较。D-ADH的当前结果与文献数据相结合，证实醛氧化（表现为歧化）是乙醇脱氢酶的普遍特性，在酒精代谢和醛解毒中具有潜在的生理重要性。
