Estonius M, Karlsson C, Fox E A, Höög J O, Holmquist B, Vallee B L, Davidson W S, Jörnvall H
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Eur J Biochem. 1990 Dec 12;194(2):593-602. doi: 10.1111/j.1432-1033.1990.tb15657.x.
The major ethanol-active form of chicken liver alcohol dehydrogenase was characterized. The primary structure was determined by peptide analysis and, to a large part, was also deduced by cDNA analysis of a near full-length cDNA clone. The latter was detected by screening of a chicken liver cDNA library with antibodies raised against the purified dehydrogenase. The structure shows that the avian enzyme exhibits characteristics of the complex mammalian alcohol dehydrogenase system, tracing its origin and divergence, and allowing functional correlations. The chicken protein analyzed proves to be a class I alcohol dehydrogenase, with 74% residue identity to gamma chains of the human enzyme, a Km for ethanol of 0.5 mM and a Ki for 4-methyl pyrazole of 2.5 microM. Relationships to the other two classes are non-identical; residue exchanges towards the human classes increase in the order I less than III less than II, and human/chicken differences are less than inter-class differences. Consequently, the origins of the classes are more distant than the avian/mammalian separation. They reflect duplicatory events separated in time, and the lines that lead to present-day classes I and II deviate early. Integrated with the data for the quail enzyme, the structure of the chicken protein shows that within the avian enzymes the degree of variation is comparable to that within the mammalian class I enzymes, which are more variable than the class III forms. The coenzyme-binding and substrate-binding residues of this chicken alcohol dehydrogenase are largely identical to those in the mammalian class I counterparts. However, the subunit-interacting areas are more variable and suggest some relationships of the avian enzyme with both class I and III mammalian forms. One of the residues, Gly260 (mammalian class I numbering system), previously considered characteristic of all alcohol dehydrogenases, is replaced by Gln.
对鸡肝乙醇脱氢酶的主要乙醇活性形式进行了表征。通过肽分析确定了其一级结构，并且在很大程度上也通过对一个近乎全长的cDNA克隆进行cDNA分析推导得出。后者是通过用针对纯化的脱氢酶产生的抗体筛选鸡肝cDNA文库而检测到的。该结构表明，禽类酶具有复杂的哺乳动物乙醇脱氢酶系统的特征，追溯其起源和分歧，并实现功能关联。所分析的鸡蛋白被证明是I类乙醇脱氢酶，与人酶的γ链具有74%的残基同一性，对乙醇的Km为0.5 mM，对4-甲基吡唑的Ki为2.5 μM。与其他两类的关系并不相同；与人的类别相比，残基交换按I＜III＜II的顺序增加，并且人与鸡的差异小于类间差异。因此，这些类别的起源比禽类/哺乳动物的分化更为久远。它们反映了在时间上分开的复制事件，并且导致当今I类和II类的谱系很早就出现了分歧。结合鹌鹑酶的数据，鸡蛋白的结构表明，在禽类酶中，变异程度与哺乳动物I类酶中的变异程度相当，而I类酶比III类形式更具变异性。这种鸡乙醇脱氢酶的辅酶结合和底物结合残基在很大程度上与哺乳动物I类对应物中的残基相同。然而，亚基相互作用区域更具变异性，表明禽类酶与哺乳动物I类和III类形式都存在一些关系。其中一个残基，Gly260（哺乳动物I类编号系统），以前被认为是所有乙醇脱氢酶的特征，被Gln取代。
