Kaaber Brendskag M, McKinley-McKee J S, Winberg J O
Department of Biochemistry, Institute of Medical Biology, University of Tromso, 9037, Tromso, Norway.
Biochim Biophys Acta. 1999 Apr 12;1431(1):74-86. doi: 10.1016/s0167-4838(99)00028-x.
The alcohol dehydrogenase (ADH) from Drosophila lebanonensis shows 82% positional identity to the alcohol dehydrogenases from Drosophila melanogaster. These insect ADHs belong to the short-chain dehydrogenase/reductase family which lack metal ions in their active site. In this family, it appears that the function of zinc in medium chain dehydrogenases has been replaced by three amino acids, Ser138, Tyr151 and Lys155. The present work on D. lebanonensis ADH has been performed in order to obtain information about reaction mechanism, and possible differences in topology and electrostatic properties in the vicinity of the catalytic residues in ADHs from various species of Drosophila. Thus the pH dependence of various kinetic coefficients has been studied. Both in the oxidation of alcohols and in the reduction of aldehydes, the reaction mechanism of D. lebanonensis ADH in the pH 6-10 region was consistent with a compulsory ordered pathway, with the coenzymes as the outer substrates. Over the entire pH region, the rate limiting step for the oxidation of secondary alcohols such as propan-2-ol was the release of the coenzyme product from the enzyme-NADH complex. In the oxidation of ethanol at least two steps were rate limiting, the hydride transfer step and the dissociation of NADH from the binary enzyme-NADH product complex. In the reduction of acetaldehyde, the rate limiting step was the dissociation of NAD+ from the binary enzyme-NAD+ product complex. The pH dependences of the kon velocity curves for the two coenzymes were the opposite of each other, i.e. kon increased for NAD+ and decreased for NADH with increasing pH. The two curves appeared complex and the kon velocity for the two coenzymes seemed to be regulated by several groups in the free enzyme. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD+ complex, with a pKa value of 7.1. The kon velocity for acetaldehyde was pH independent and showed that in the enzyme-NADH complex, the pKa value of the catalytic residue must be above 10. The koff velocity of NAD+ appeared to be partly regulated by the catalytic residue, and protonation resulted in an increased dissociation rate. The koff velocity for NADH and the hydride transfer step was pH independent. In D. lebanonensis ADH, the pKa value of the catalytic residue was 0.5 pH units lower than in the ADHS alleloenzyme from D. melanogaster. Thus it can be concluded that while most of the topology of the active site is mainly conserved in these two distantly related enzymes, the microenvironment and electrostatic properties around the catalytic residues differ.
黎巴嫩果蝇的乙醇脱氢酶（ADH）与黑腹果蝇的乙醇脱氢酶在位置上有82%的一致性。这些昆虫ADH属于短链脱氢酶/还原酶家族，其活性位点不含金属离子。在这个家族中，中链脱氢酶中锌的功能似乎已被三个氨基酸Ser138、Tyr151和Lys155所取代。对黎巴嫩果蝇ADH的当前研究是为了获取有关反应机制以及不同果蝇物种ADH催化残基附近拓扑结构和静电性质可能存在的差异的信息。因此，研究了各种动力学系数的pH依赖性。在醇的氧化和醛的还原过程中，黎巴嫩果蝇ADH在pH 6 - 10区域的反应机制与强制有序途径一致，辅酶作为外部底物。在整个pH区域，仲醇（如丙 - 2 - 醇）氧化的限速步骤是辅酶产物从酶 - NADH复合物中的释放。在乙醇氧化过程中，至少有两个步骤是限速的，即氢化物转移步骤以及NADH从二元酶 - NADH产物复合物中的解离。在乙醛还原过程中，限速步骤是NAD⁺从二元酶 - NAD⁺产物复合物中的解离。两种辅酶的kon速度曲线的pH依赖性彼此相反，即随着pH升高，NAD⁺的kon增加而NADH的kon降低。这两条曲线看起来很复杂，两种辅酶的kon速度似乎受游离酶中的几个基团调节。乙醇和乙醇竞争性抑制剂吡唑的kon速度随pH升高，并且通过二元酶 - NAD⁺复合物中单个基团的电离来调节，其pKa值为7.1。乙醛的kon速度与pH无关，表明在酶 - NADH复合物中，催化残基的pKa值必须高于10。NAD⁺的koff速度似乎部分受催化残基调节，质子化导致解离速率增加。NADH的koff速度和氢化物转移步骤与pH无关。在黎巴嫩果蝇ADH中，催化残基的pKa值比黑腹果蝇的ADHS等位酶低0.5个pH单位。因此可以得出结论，虽然这两种远缘相关酶的活性位点的大部分拓扑结构主要是保守的，但催化残基周围的微环境和静电性质有所不同。
