Strasser F, Dey J, Eftink M R, Plapp B V
Department of Biochemistry, The University of Iowa, Iowa City, Iowa, 52242, USA.
Arch Biochem Biophys. 1998 Oct 15;358(2):369-76. doi: 10.1006/abbi.1998.0882.
Horse liver alcohol dehydrogenase contains two tryptophan residues per subunit, Trp-15 on the surface of the catalytic domain and Trp-314 buried in the interface between the subunits of the dimer. We studied the contributions of the tryptophans to fluorescence and catalytic dynamics by substituting Trp-314 with a leucine residue and making two compensatory mutations that were required to obtain a stable protein, leading to the triple mutant M303F-L308I-W314L enzyme. The substitutions increased by two- to sixfold the turnover numbers for ethanol oxidation, acetaldehyde reduction, and the dissociation constants of the coenzymes. The rate of the exponential burst phase for the transient oxidation of ethanol increased slightly, but the rate of dissociation of the enzyme-NADH complex still limited turnover of ethanol, as for wild-type enzyme. The three substitutions at the dimer interface apparently activate the enzyme by allowing more rapid conformational changes that accompany coenzyme binding, probably due to movement of the loop containing residues 293 to 298. The emission spectrum of M303F-L308I-W314L enzyme, which contains Trp-15, was redshifted compared to wild-type enzyme. Time-resolved fluorescence measurements with the triple mutant show that the decay of Trp-15 is dominated by a approximately 7-ns component. In the mutant enzyme with Trp-15 substituted with phenylalanine, the decay of Trp-314 is dominated by a approximately 4-ns component. Solute quenching data for wild-type enzyme and the mutants show that only Trp-15 is exposed to iodide and acrylamide, whereas Trp-314 is inaccessible. The luminescence properties of the tryptophan residues in the mutated enzymes are consistent with conclusions from studies of the wild-type enzyme [M. R. Eftink, 1992, Adv. Biophys. Chem. 2, 81-114].
马肝醇脱氢酶每个亚基含有两个色氨酸残基，催化结构域表面的Trp-15和二聚体亚基之间界面处埋藏的Trp-314。我们通过用亮氨酸残基取代Trp-314并进行两个获得稳定蛋白所需的补偿性突变来研究色氨酸对荧光和催化动力学的贡献，从而得到三重突变体M303F-L308I-W314L酶。这些取代使乙醇氧化、乙醛还原的周转数以及辅酶的解离常数增加了两到六倍。乙醇瞬时氧化的指数爆发相速率略有增加，但酶-NADH复合物的解离速率仍然限制了乙醇的周转，这与野生型酶的情况相同。二聚体界面处的三个取代显然通过允许伴随辅酶结合的更快构象变化来激活酶，这可能是由于包含残基293至298的环的移动。含有Trp-15的M303F-L308I-W314L酶的发射光谱与野生型酶相比发生了红移。对三重突变体的时间分辨荧光测量表明，Trp-15的衰减主要由一个约7纳秒的成分主导。在将Trp-15替换为苯丙氨酸的突变酶中，Trp-314的衰减主要由一个约4纳秒的成分主导。野生型酶和突变体的溶质猝灭数据表明，只有Trp-15暴露于碘化物和丙烯酰胺，而Trp-314无法接近。突变酶中色氨酸残基的发光特性与野生型酶的研究结论一致[M.R.埃夫廷克，1992，《生物物理化学进展》2，81-114]。
