Ohama H, Sugiura N, Tanaka F, Yagi K
Biochemistry. 1977 Jan 11;16(1):126-31. doi: 10.1021/bi00620a021.
The absorption spectrum of D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) was significantly perturbed by various alcohols; typical fine structures were observed in the visible absorption bands, accompanied by blue shifts of the peaks. Both fluorescence intensity and fluorescence polarization were increased upon the addition of alcohols, indicating that the coenzyme is not liberated from the apoenzyme but the hydrophobicity of the environment of the enzyme-bound flavin is increased. Upon the addition of alcohols, the circular dichroism of the enzyme was markedly modified in the visible and near-ultraviolet regions, while that of the apoenzyme in the near- and far-ultraviolet regions was scarcely modified, indicating a change in the interaction between the flavin coenzyme and protein. Both the apparent maximal velocity and the apparent Michaelis constant of the enzyme were increased by the addition of alcohols. The presence of alcohols tends to dissociate the dimer of this enzyme into the monomer, but the dissociation does not fully explain the increase in the maximal velocity of the enzyme by alcohols, because the increase in the maximal velocity caused by alcohols is larger than that expected from the dissociation. Since the rate of formation of the purple intermediate was decreased by alcohols in both the dimer and the monomer, the increase in the maximal velocity could be ascribed to an increase in the rate of dissociation of the enzyme-product complex. This increase could be ascribed to the protein conformational change, which is probably provoked by combination of alcohols with the enzyme at a locus other than that for substrate binding.
D-氨基酸氧化酶（D-氨基酸：氧氧化还原酶（脱氨基），EC 1.4.3.3）的吸收光谱受到各种醇类的显著干扰；在可见吸收带中观察到典型的精细结构，同时伴有峰的蓝移。加入醇类后，荧光强度和荧光偏振均增加，这表明辅酶并未从脱辅基酶中释放出来，而是酶结合黄素环境的疏水性增加。加入醇类后，酶在可见光和近紫外区域的圆二色性发生了显著改变，而脱辅基酶在近紫外和远紫外区域的圆二色性几乎没有改变，这表明黄素辅酶与蛋白质之间的相互作用发生了变化。加入醇类后，该酶的表观最大反应速度和表观米氏常数均增加。醇类的存在倾向于使该酶的二聚体解离为单体，但这种解离并不能完全解释醇类使酶的最大反应速度增加的原因，因为醇类引起的最大反应速度增加幅度大于由解离所预期的幅度。由于醇类使二聚体和单体中紫色中间体的形成速率均降低，因此最大反应速度的增加可归因于酶-产物复合物解离速率的增加。这种增加可能归因于蛋白质构象变化，这可能是由醇类与酶在底物结合位点以外的位点结合所引发的。
