Kitson T M
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Biochem J. 1989 Jan 15;257(2):585-90. doi: 10.1042/bj2570585.
Cytoplasmic aldehyde dehydrogenase may be modified by reaction with p-nitrophenyl dimethylcarbamate to give a stable E-X-CO-NMe2 species that is an analogue of the usual labile acyl-enzyme involved in the enzyme's reactions. [X is derived from an enzymic nucleophilic group.] This species still contains the tightly bound NADH that is present in the native enzyme. When further NADH binds to E-X-CO-NMe2 its fluorescence is enhanced over 4 times more than when it binds to unmodified enzyme; this fluorescence is completely unaffected by high propionaldehyde concentration and only slightly affected by p-nitrobenzaldehyde. The modified species has 1.0 NADH binding site in the absence of Mg2+ and 1.67 sites in its presence. The rate of dissociation of E-X-CO-NMe2.NADH is biphasic (k 3.4 and 1.8 min-1) and is considerably lower than that of E.NADH; the presence of Mg2+ slows the process even more (k 0.47 and 0.37 min-1). The implications of these studies towards a greater understanding of the nature of aldehyde dehydrogenase-catalysed reactions are discussed.
细胞质醛脱氢酶可通过与对硝基苯基二甲基氨基甲酸酯反应进行修饰，生成稳定的E-X-CO-NMe2物种，该物种是参与该酶反应的常见不稳定酰基酶的类似物。[X源自酶的亲核基团。]该物种仍含有天然酶中存在的紧密结合的NADH。当进一步的NADH与E-X-CO-NMe2结合时，其荧光增强超过4倍，比与未修饰的酶结合时增强得更多；这种荧光完全不受高浓度丙醛的影响，仅受对硝基苯甲醛的轻微影响。在不存在Mg2+的情况下，修饰后的物种有1.0个NADH结合位点，在存在Mg2+的情况下有1.67个位点。E-X-CO-NMe2.NADH的解离速率是双相的（k分别为3.4和1.8 min-1），且远低于E.NADH的解离速率；Mg2+的存在使该过程进一步减慢（k分别为0.47和0.37 min-1）。讨论了这些研究对更深入理解醛脱氢酶催化反应性质的意义。
