PURIFICATION AND STEADY-STATE KINETIC CHARACTERIZATION OF HUMAN-LIVER BETA-3-BETA-3 ALCOHOL-DEHYDROGENASE
PURIFICATION AND STEADY-STATE KINETIC CHARACTERIZATION OF HUMAN-LIVER BETA-3-BETA-3 ALCOHOL-DEHYDROGENASE
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DOI：
10.1021/bi00443a005
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发表时间：
1989-08-22
期刊：
影响因子：
2.9
通讯作者：
BOSRON, WF
中科院分区：
文献类型：
--
作者：
BURNELL, JC;LI, TK;BOSRON, WF
人肝乙醇脱氢酶催化依赖NAD+的醇氧化。肝脏中的同工酶由五种不同的基因产生，其中两种是多态的。我们已经研究了三个.测试版..测试版。ADH2产生的同工酶是因为它们表现出非常不同的动力学性质，并且它们在不同的种族群体中出现的频率不同。在25%的美国黑人中出现的β3β3同工酶被纯化为均一种，并找到了稳定这种不稳定的同工酶的条件。底物专一性的比较。伯直链醇的同工酶表明，对于β2β2和β3β3的醇，Vmax/Km与对数辛醇/水分配系数呈正相关，而与β1β1无关。这些醇的C1或C2上的甲基取代降低了所有三种同工酶的催化效率。对于NAD+和NADH，β3β3的Km和Ki值显著高于β1β1或β2β2的值。β3β3乙醇氧化的Vmax是β1β1的90倍。对β3亚基和基因的测序表明，该多态是由于β3中的Cys-369与β1和β2中的Arg-369的单一氨基酸交换所致[Burnell等人。(1987)生物化学。生物群落。[中英文摘要]Re.Commun.146、1227-1233]。在马酒精脱氢酶和β1β1中，Arg-369的胍基被认为通过与焦磷酸氧基之一结合来稳定NAD(H)酶复合体。因此，Cys-369在β3β3中的取代解释了其辅酶结合能力弱和活性高的原因。
Human liver alcohol dehydrogenase catalyzes the NAD+-dependent oxidation of alcohols. Isoenzymes are produced in liver by five different genes, two of which are polymorphic. We have studied the three .beta..beta. isoenzymes produced at ADH2 because they exhibit very different kinetic properties and they appear with different frequencies in different racial populations. The .beta.3.beta.3 isoenzyme which appears in 25% of black Americans was purified to homogeneity, and conditions were found to stabilize this labile isoenzyme. The comparison of substrate specificity among .beta..beta. isoenzymes for primary straight-chain alcohols indicates that there is a positive correlation between Vmax/KM and the log octanol/water partition coefficient for alcohols with .beta.2.beta.2 and .beta.3.beta.3 but not with .beta.1.beta.1. Methyl substitutions at C1 or C2 of these alcohols reduce the catalytic efficiency with all three isoenzymes. The KM and Ki values of .beta.3.beta.3 for NAD+ and NADH are substantially higher than values for .beta.1.beta.1 or .beta.2.beta.2. The Vmax of .beta.3.beta.3 for ethanol oxidation is 90 times that of .beta.1.beta.1. Sequencing of the .beta.3 subunit and gene indicates that the polymorphism results from a single amino acid exchange of Cys-369 in .beta.3 for Arg-369 in .beta.1 and .beta.2 [Burnell et al. (1987) Biochem. Biophys. Res. Commun. 146, 1227-1233]. In horse alcohol dehydrogenase and .beta.1.beta.1, the guanidino group of Arg-369 is thought to stabilize the NAD(H)-enzyme complex by bonding to one of the pyrophosphate oxygens. Thus, the substitution of Cys-369 in .beta.3.beta.3 explains its weak binding of coenzymes and high activity.