AMINO-ACIDS IMPORTANT IN ENZYME-ACTIVITY AND DIMER STABILITY FOR DROSOPHILA ALCOHOL-DEHYDROGENASE
AMINO-ACIDS IMPORTANT IN ENZYME-ACTIVITY AND DIMER STABILITY FOR DROSOPHILA ALCOHOL-DEHYDROGENASE
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DOI：
10.1042/bj3080419
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发表时间：
1995-06-01
影响因子：
4.1
通讯作者：
LEE, WR
中科院分区：
文献类型：
--
作者：
CHENEVERT, SW;FOSSETT, NG;LEE, WR
我们已经确定了果蝇醇脱氢酶（ADH）中八种甲磺酸乙酯诱导的突变体的核苷酸序列，其中六种先前由Hollocher和Place表征[（1988）Genetics 116，253-263和265-274]。这些ADH突变体中的四个含有单个氨基酸变化：甘氨酸-17变为精氨酸，甘氨酸-93变为谷氨酸，丙氨酸-159变为苏氨酸，甘氨酸-184变为天冬氨酸。虽然这些突变体是无活性的，但三种突变体（Gly 17 Arg、Gly 93 Glu和Gly 184 Asp）与野生型ADH形成稳定的同源二聚体以及异源二聚体，其中野生型ADH亚基保留完全的酶活性[Hollocher和Place（1988）Genetics 116，265-274]。有趣的是，Ala 159 Thr突变体与野生型ADH不形成稳定的同源二聚体或异源二聚体，这表明丙氨酸-159在稳定ADH二聚体方面是重要的。使用细菌20 β-羟基类固醇脱氢酶和大鼠二氢蝶啶还原酶作为模板，在ADH的三维模型方面分析突变。该模型表明甘氨酸-17和甘氨酸-93中的突变影响NAD（+）的结合。它还表明，丙氨酸-159是ADH的二聚体界面上的疏水锚的一部分。用苏氨酸取代丙氨酸-159，苏氨酸具有更大的侧链并且可以与水形成氢键，可能会降低疏水相互作用的强度。三维模型显示甘氨酸-184靠近底物结合位点。用天冬氨酸取代甘氨酸-184可能会改变苏氨酸-186的位置，我们认为苏氨酸-186与NAD（+）的羧酰胺部分形成氢键。此外，天冬氨酸侧链上的负电荷可与底物和/或底物结合位点中的残基相互作用。这些突变提供了有关ADH催化和二聚体稳定性的信息，这也可能有助于理解同源性类固醇脱氢酶，包括人17 β-羟基类固醇、11 β-羟基类固醇和15-羟基前列腺素脱氢酶。
We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers, The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD(+). It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD(+). Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.