Ditlow C C, Holmquist B, Morelock M M, Vallee B L
Biochemistry. 1984 Dec 18;23(26):6363-8. doi: 10.1021/bi00321a012.
Homogeneous class II alcohol dehydrogenase (pi-ADH) has been isolated from human liver homogenates by chromatography on DE-52 cellulose, 4-[3-[N-(6-amino-caproyl)amino]propyl]pyrazole-Sepharose, SP-Sephadex C-50, and agarose-hexane-AMP, yielding an enzyme that has a significantly higher specific activity and is markedly more stable than that isolated by an earlier procedure. pi-ADH is composed of two identical 40 000-dalton subunits, contains 4 mol of zinc/dimer, and is readily inhibited by metal-chelating agents. The purified enzyme binds two molecules of coenzyme per dimer, exhibits an absorption maximum at 280 nm, epsilon 280 = 57 000, and exhibits an isoelectric point of 8.6. The class II isozyme catalyzes the oxidation of a variety of alcohols with Km values ranging from 7 microM to 560 mM and with kcat values from 32 min-1 to 600 min-1 and demonstrates a preference for hydrophobic substrates. The kcat/Km ratio for ethanol oxidation exhibits a pH maximum at 10.4.
通过在DE - 52纤维素、4 - [3 - [N - (6 - 氨基己酰基)氨基]丙基]吡唑 - 琼脂糖、SP - 葡聚糖凝胶C - 50和琼脂糖 - 己烷 - AMP上进行色谱分离，从人肝匀浆中分离出了均一的II类乙醇脱氢酶（pi - ADH），得到的一种酶具有显著更高的比活性，并且比通过早期方法分离的酶明显更稳定。pi - ADH由两个相同的40000道尔顿亚基组成，每二聚体含有4摩尔锌，并且容易被金属螯合剂抑制。纯化后的酶每个二聚体结合两个辅酶分子，在280nm处有最大吸收峰，ε280 = 57000，等电点为8.6。II类同工酶催化多种醇的氧化，其Km值范围为7μM至560mM，kcat值范围为32min⁻¹至600min⁻¹，并且对疏水性底物表现出偏好。乙醇氧化的kcat/Km比值在pH为10.4时达到最大值。
