Holmquist B, Moulis J M, Engeland K, Vallee B L
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1993 May 18;32(19):5139-44. doi: 10.1021/bi00070a024.
Modification of class III alcohol dehydrogenase (chi chi-ADH) with phenylglyoxal eliminates fatty acid activation by pentanoate and octanoate and concomitantly increases specific activity toward ethanol and 3-methylcrotyl alcohol 2-3-fold. In contrast, chemical modification decreases activity toward S-(hydroxymethyl)glutathione (FDH activity) and 12-hydroxydodecanoic acid by increasing Km, pointing to a role for arginine in binding anionic substrates. Modification with [7-14C]phenylglyoxal indicates that only one arginine residue per subunit is modified. Sequence analysis of tryptic peptides indicates that Arg-115 is modified. Site-directed mutation of this residue to alanine eliminates both fatty acid activation and FDH activity, thus confirming the identity of the modified residue and its function. These results account in part for the unique specificity of chi chi-ADH relative to other human ADH isozymes.
用苯乙二醛修饰Ⅲ类乙醇脱氢酶（chi chi-ADH）可消除戊酸和辛酸对脂肪酸的激活作用，并同时使对乙醇和3-甲基巴豆醇的比活性提高2至3倍。相比之下，化学修饰通过增加米氏常数（Km）降低了对S-（羟甲基）谷胱甘肽（FDH活性）和12-羟基十二烷酸的活性，表明精氨酸在结合阴离子底物中起作用。用[7-14C]苯乙二醛修饰表明每个亚基只有一个精氨酸残基被修饰。胰蛋白酶肽段的序列分析表明被修饰的是精氨酸-115。将该残基定点突变为丙氨酸可消除脂肪酸激活作用和FDH活性，从而证实了被修饰残基的身份及其功能。这些结果部分解释了chi chi-ADH相对于其他人类ADH同工酶的独特特异性。
