Wagner F W, Burger A R, Vallee B L
Biochemistry. 1983 Apr 12;22(8):1857-63. doi: 10.1021/bi00277a018.
Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.
通过在二乙氨基乙基纤维素、4-[3-[N-(6-氨基己酰基)-氨基]丙基]吡唑-琼脂糖和羧甲基纤维素上对人肝匀浆进行色谱分析，分离出了I类乙醇脱氢酶（ADH）同工酶。纯化了8种不同亚基组成（αγ2、γ2γ2、αγ1、αβ1、β1γ2、γ1γ1、β1γ1和β1β1）的同工酶，并在pH 10.0条件下，以乙醇、乙二醇、甲醇、苯甲醇、辛醇、环己醇和16-羟基十六烷酸作为底物测定了它们的活性。除β1β1-ADH外，所有同工酶对乙醇氧化的米氏常数（Km）和催化常数（kcat）值相似，但对其他醇类的氧化值则有显著差异。β1β1-ADH的kcat值不变（约10 min-1），且比所研究的任何其他I类同工酶的kcat值低得多（5-15倍）。甲醇和乙二醇的Km值比乙醇的Km值大5至100倍，具体取决于同工酶，而苯甲醇、辛醇和16-羟基十六烷酸的Km值通常比乙醇的Km值低100-1000倍。对于所研究的除甲醇和乙二醇外的所有醇类，同二聚体β1β1的kcat/Km值最低；所有同工酶作用于所有醇类时，kcat值相对恒定，因此，特异性主要体现在Km值上。所检测的所有酶的Km值和kcat/Km值表明，短链醇是最差的底物，而带有庞大取代基的醇是较好的底物。异二聚体动力学参数的实验值偏离了其亲本同二聚体计算出的平均值，因此，无法根据后者的行为进行预测。因此，ADH的异二聚体和同二聚体同工酶对给定底物的特异性是各自的特征。对于作为人类酶主要形式的所有I类同工酶而言，乙醇被证明是所检测的“最差”底物之一。基于动力学标准，所研究的I类同工酶中没有一种以表明对该醇有酶促偏好的方式氧化乙醇。
