Karlović D, Amiguet P, Bonner F J, Luisi P L
Eur J Biochem. 1976 Jul 1;66(2):277-84. doi: 10.1111/j.1432-1033.1976.tb10517.x.
Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.
已研究了酵母醇脱氢酶及其辅酶复合物的校正荧光性质随温度的变化情况。通过跟踪NADH荧光增强或蛋白质荧光猝灭，获得了NAD和NADH二元及三元复合物的解离常数。发现吡唑的存在使NAD对该酶的亲和力增加约100倍。三元酶-NAD-吡唑复合物的形成伴随着紫外吸收性质的巨大变化，在290nm区域出现一条新带。三元酶-NADH-乙酰胺复合物的形成也伴随着显著的光学变化。讨论了辅酶结合后蛋白质荧光猝灭的可能原因，认为辅酶诱导的构象变化可能导致这种情况。基于解离常数随温度的变化，评估了与NAD和NADH结合相关的热力学参数。将酵母醇脱氢酶二元和三元复合物的光学和热力学性质与马肝醇脱氢酶的类似性质进行了比较。
