MacKerell A D, Pietruszko R
Biochim Biophys Acta. 1987 Feb 25;911(3):306-17. doi: 10.1016/0167-4838(87)90071-9.
Employing 3,4-dihydroxyphenylacetaldehyde (dopal) as a substrate for human aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) in anaerobic conditions, inactivation of both cytoplasmic E1 and mitochondrial E2 isozymes during catalysis has been observed. Incorporation of 14C-labelled dopal has been demonstrated by retention of label following denaturation and exhaustive dialysis and by peptide mapping following tryptic digestion. Incorporation of label gave linear plots vs. activity remaining with up to two molecules incorporated per molecule of enzyme and 30% activity remaining. Further incorporation (up to 16 molecules) occurred, but was non-linear when plotted vs. activity remaining. Protection against activity loss during incorporation of the first two molecules was afforded by NAD, NADH, chloral, and by chloral and NAD together, the last being the most effective. Saturation kinetics gave y-axis intercepts, suggesting interaction at a specific point on the enzyme surface. The Ki value from saturation kinetics was the same as that from the slope replot in catalytic reaction. Peptide mapping of tryptic digests showed that a single peptide was labelled, confirming specificity of interaction. Even in the absence of complete inactivation, the results suggest that reaction with the first two molecules occurs at some point on the enzyme surface important for enzyme activity. The possibility of such a reaction occurring in vivo is discussed.
在厌氧条件下，以3,4 - 二羟基苯乙醛（多巴醛）作为人醛脱氢酶（醛：NAD⁺氧化还原酶，EC 1.2.1.3）的底物时，已观察到催化过程中细胞质E1和线粒体E2同工酶均失活。变性和彻底透析后标记的保留以及胰蛋白酶消化后的肽图谱分析均证实了¹⁴C标记的多巴醛的掺入。标记掺入与剩余活性呈线性关系，每个酶分子最多掺入两个分子且剩余30%的活性。进一步的掺入（最多16个分子）也会发生，但与剩余活性作图时呈非线性。NAD、NADH、三氯乙醛以及三氯乙醛和NAD共同作用可防止在前两个分子掺入过程中的活性损失，其中最后一种最为有效。饱和动力学给出了y轴截距，表明在酶表面的特定点发生了相互作用。饱和动力学的Ki值与催化反应中斜率重作图的Ki值相同。胰蛋白酶消化产物的肽图谱分析表明只有一个肽被标记，证实了相互作用的特异性。即使在没有完全失活的情况下，结果也表明与前两个分子的反应发生在酶表面对酶活性很重要的某个点上。本文还讨论了这种反应在体内发生的可能性。
