Wang X, Sheikh S, Saigal D, Robinson L, Weiner H
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
J Biol Chem. 1996 Dec 6;271(49):31172-8. doi: 10.1074/jbc.271.49.31172.
About 50% of the Oriental population have less liver mitochondrial aldehyde dehydrogenase (ALDH2) activity than do other people. It was found that they possessed an enzyme with a lysine at position 487 (E487K) instead of glutamate (Glu487). We previously found that the Km for NAD of recombinant human and rat E487K enzymes increased more than 150-fold (Farrés, J., Wang X., Takahashi, K., Cunningham, S. J. , Wang, T.T., and Weiner, H (1994) J. Biol. Chem. 269, 13854-13860). Many aldehyde dehydrogenase-deficient people were found to be heterozygous when genotyped for ALDH2. In this study liver tissue from heterozygous people was analyzed and found to possess mRNAs for both the glutamate and the lysine subunits. Western blot analysis showed that the glutamate subunit was present. The cDNAs for Glu487 and E487K were coexpressed on one plasmid in Escherichia coli, and the enzyme forms were separated from each other by isoelectric focusing to show that heterotetramers were formed. Only one Km value for NAD could be measured with the purified heterotetrameric enzyme that possessed just 16-18% activity of the glutamate homotetrameric enzyme. The E487K homotetramers had 8% specific activity of the Glu487 enzyme. There was no pre-steady state burst of NADH formation with the heterotetramer, a property found with the glutamate enzyme. Similar results were found for the coexpressed rat liver enzyme, except that a higher specific activity, 48%, was obtained. Thus, we conclude that presence of the lysine subunit altered the activity of the glutamate subunit in the heterotetramer to make it function more like an E487K enzyme.
约50%的东方人群肝脏线粒体乙醛脱氢酶（ALDH2）活性低于其他人。研究发现，他们拥有一种在487位为赖氨酸（E487K）而非谷氨酸（Glu487）的酶。我们之前发现，重组人及大鼠E487K酶对NAD的Km值增加了150倍以上（法雷斯，J.，王X.，高桥K.，坎宁安，S.J.，王T.T.，以及韦纳，H（1994年）《生物化学杂志》269卷，13854 - 13860页）。许多乙醛脱氢酶缺陷人群在进行ALDH2基因分型时被发现为杂合子。在本研究中，对杂合子人群的肝脏组织进行了分析，发现其同时拥有谷氨酸和赖氨酸亚基的mRNA。蛋白质印迹分析表明存在谷氨酸亚基。将Glu487和E487K的cDNA在大肠杆菌的一个质粒上共表达，并通过等电聚焦将酶形式彼此分离，以显示形成了异源四聚体。用纯化的异源四聚体酶只能测得一个NAD的Km值，该酶的活性仅为谷氨酸同型四聚体酶的16 - 18%。E487K同型四聚体的比活性为Glu487酶的8%。异源四聚体没有谷氨酸酶所具有的NADH形成的稳态前爆发特性。对共表达的大鼠肝脏酶也得到了类似结果，只是比活性更高，为48%。因此，我们得出结论，赖氨酸亚基的存在改变了异源四聚体中谷氨酸亚基的活性，使其功能更像E487K酶。
