Wolfe J K, Weidig C F, Halvorson H R, Shore J D
J Biol Chem. 1977 Jan 25;252(2):433-6.
The quenching of liver alcohol dehydrogenase protein fluorescence at alkaline pH indicates two conformational states of the enzyme with a pKa of 9.8+/-0.2, shifted to 10.6+/-0.2 in D2O. NAD+ and 2-p-toluidinonaphthalene-6-sulfonate, a fluorescent probe competitive with coenzyme, bind to the acid conformation of the enzyme. The pKa of the protein-fluorescence quenching curve is shifted toward 7.6 in the presence of NAD+, and the ternary complex formation with NAD+ and trifluoroethanol results in a pH-independent maximal quench. At pH (pD) 10.5, the rate constant for NAD+ binding was 2.6 times faster in D2O2 than in H2O due to the shift of the pKa. Based on these results, a scheme has been proposed in which the state of protonation of an enzyme functional group with a pKa of 9.8 controls the conformational state of the enzyme. NAD+ binds to the acid conformation and subsequently causes another conformational change resulting in the perturbation of the pKa to 7.6. Alcohol then binds to the unprotonated form of the functional group with a pKa of 7.6 in the binary enzyme-NAD+ complex and converts the enzyme to the alkaline conformation. Thus, at neutral pH liver alcohol dehydrogenase undergoes two conformational changes en route to the ternary complex in which hydride transfer occurs.
在碱性pH条件下，肝脏乙醇脱氢酶蛋白荧光的猝灭表明该酶存在两种构象状态，其pKa为9.8±0.2，在重水中则变为10.6±0.2。NAD⁺和2-对甲苯胺基萘-6-磺酸盐（一种与辅酶竞争的荧光探针）与酶的酸性构象结合。在NAD⁺存在下，蛋白质荧光猝灭曲线的pKa向7.6移动，并且与NAD⁺和三氟乙醇形成的三元复合物导致与pH无关的最大猝灭。在pH（pD）10.5时，由于pKa的移动，NAD⁺结合的速率常数在重水中比在水中快2.6倍。基于这些结果，提出了一种机制，其中pKa为9.8的酶功能基团的质子化状态控制酶的构象状态。NAD⁺与酸性构象结合，随后引起另一种构象变化，导致pKa扰动至7.6。然后乙醇与二元酶-NAD⁺复合物中pKa为7.6的功能基团的未质子化形式结合，并将酶转化为碱性构象。因此，在中性pH条件下，肝脏乙醇脱氢酶在形成三元复合物（其中发生氢化物转移）的过程中经历两种构象变化。
