EXPRESSION AND KINETIC CHARACTERIZATION OF RECOMBINANT HUMAN STOMACH ALCOHOL-DEHYDROGENASE - ACTIVE-SITE AMINO-ACID-SEQUENCE EXPLAINS SUBSTRATE-SPECIFICITY COMPARED WITH LIVER ISOZYMES
EXPRESSION AND KINETIC CHARACTERIZATION OF RECOMBINANT HUMAN STOMACH ALCOHOL-DEHYDROGENASE - ACTIVE-SITE AMINO-ACID-SEQUENCE EXPLAINS SUBSTRATE-SPECIFICITY COMPARED WITH LIVER ISOZYMES
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DOI：
10.1074/jbc.270.8.3625
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发表时间：
1995-02-24
影响因子：
4.8
通讯作者：
LI, TK
中科院分区：
文献类型：
--
作者：
KEDISHVILI, NY;BOSRON, WF;LI, TK
从人胃cDNA文库中分离得到人IV类乙醇脱氢酶（sigma-ADH）的全长1966个碱基对的克隆。由该cDNA编码的373个氨基酸的σ-ADH在大肠杆菌中表达，重组酶在pH 7.5和25 ℃下对乙醇氧化的比活性（由NADH结合的活性位点滴定计算）为92 +/-9单位/mg，重组σ-ADH氧化伯醇的催化效率（k（cat）/K-M）的动力学分析表明宽底物特异性，重组人sigma-ADH对全反式视黄醇氧化为全反式视黄醛具有高催化效率。该途径在转录调节剂全反式视黄酸的合成中是重要的。仲醇和3 β-羟基类固醇对σ-ADH无活性或以非常低的效率被氧化。α-ADH对乙醇的K-M为25 mM，并且对伯直链醇的K-M随着链长的增加而显著降低。在人IV类（σ）和人I类（β）醇脱氢酶之间的醇结合位点中存在重要的氨基酸差异，这似乎解释了相对于β（1）-ADH，σ-ADH对全反式-视黄醇的高催化效率、对乙醇的高k（cat）和对仲醇的低催化效率。模拟人β（1）-ADH结构中全反式视黄醇的结合表明视黄醇与活性位点锌的配位受到位于醇结合位点入口处的残基114至120的环的阻碍，在人σ-ADH中Gly-117的缺失和Leu对大体积Tyr-110的取代似乎有助于视黄醇进入活性位点锌。
A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (sigma-ADH) was isolated from a human stomach cDNA library. The 373-amino acid sigma-ADH encoded by this cDNA was expressed in Escherichia coli, The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25 degrees C, calculated from active-site titration of NADH binding, was 92 +/- 9 units/mg, Kinetic analysis of the catalytic efficiency (k(cat)/K-M) of recombinant sigma-ADH for oxidation of primary alcohols indicated broad substrate specificity, Recombinant human sigma-ADH exhibited high catalytic efficiency for oxidation of all-trans-retinol to all-trans-retinal. This pathway is important in the synthesis of the transcriptional regulator all-trans-retinoic acid, Secondary alcohols and 3 beta-hydroxysteroids were inactive with sigma-ADH or were oxidized with very low efficiency, The K-M of a-ADH for ethanol was 25 mM, and the K-M for primary straight chain alcohols decreased substantially as chain length increased. There are important amino acid differences in the alcohol-binding site between the human class IV (sigma) and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high k(cat) for ethanol, and the low catalytic efficiency for secondary alcohols of sigma-ADH relative to beta(1)-ADH, For example, modeling the binding of all-trans-retinol in the human beta(1)-ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site, The deletion of Gly-117 in human sigma-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.