Blatter E E, Tasayco M L, Prestwich G, Pietruszko R
Center of Alcohol Studies, Rutgers University, Piscataway, NJ 08855-0969.
Biochem J. 1990 Dec 1;272(2):351-8. doi: 10.1042/bj2720351.
A major component of the sex pheromone from the tobacco budworm moth Heliothis virescens is a C16 straight-chain aldehyde with a single unsaturation at the eleventh position. The sex pheromones are inactivated when metabolized to their corresponding acids by insect aldehyde dehydrogenase. During this investigation it was demonstrated that the C16 aldehyde is a good substrate for human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 with Km and Kcat. values at pH 7.0 of 2 microM and 0.4 mumol of NADH/min per mg and of 0.6 microM and 0.24 mumol of NADH/min per mg respectively. A vinyl ketone analogue of the pheromone inhibited insect pheromone metabolism; it also inactivated human aldehyde dehydrogenase. Total inactivation of both isoenzymes was achieved at stoichiometric (equal or less than the subunit number) concentrations of vinyl ketone, incorporating 2.1-2.6 molecules/molecule of enzyme. Substrate protection was observed in the presence of the parent aldehyde and 5'-AMP. Peptide maps of tryptic digests of the E2 isoenzyme modified with 3H-labelled vinyl ketone showed that incorporation occurred into a single peptide peak. The labelled peptide of E2 isoenzyme was further purified on h.p.l.c. and sequenced. The label was incorporated into cysteine-302 in the primary structure of E2 isoenzyme, thus indicating that cysteine-302 is located in the aldehyde substrate area of the active site of aldehyde dehydrogenase. Affinity labelling of aldehyde dehydrogenase with vinyl ketones may prove to be of general utility in biochemical studies of these enzymes.
烟草夜蛾（烟芽夜蛾）性信息素的一个主要成分是一种C16直链醛，其在第十一位有一个单不饱和键。当昆虫醛脱氢酶将性信息素代谢为相应的酸时，性信息素就会失活。在本研究中，已证明该C16醛是人类醛脱氢酶（EC 1.2.1.3）同工酶E1和E2的良好底物，在pH 7.0时，其Km和Kcat值分别为2微摩尔和每毫克0.4微摩尔NADH/分钟，以及0.6微摩尔和每毫克0.24微摩尔NADH/分钟。该性信息素的乙烯基酮类似物抑制昆虫性信息素代谢；它也使人类醛脱氢酶失活。在化学计量（等于或小于亚基数量）浓度的乙烯基酮存在下，两种同工酶都完全失活，每个酶分子结合2.1 - 2.6个分子。在亲本醛和5'-AMP存在下观察到底物保护作用。用3H标记的乙烯基酮修饰的E2同工酶胰蛋白酶消化物的肽图显示，标记掺入到一个单一的肽峰中。E2同工酶的标记肽在高效液相色谱上进一步纯化并测序。标记掺入到E2同工酶一级结构中的半胱氨酸-302，因此表明半胱氨酸-302位于醛脱氢酶活性位点的醛底物区域。用乙烯基酮对醛脱氢酶进行亲和标记可能在这些酶的生化研究中具有普遍用途。
