Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue.
Chemical modification of 3 alpha,20 beta-hydroxysteroid dehydrogenase with diethyl pyrocarbonate. Evidence for an essential, highly reactive, lysyl residue.
复制标题
用焦碳酸二乙酯对 3α,20β-羟基类固醇脱氢酶进行化学修饰。
作者：
P. Pasta;G. Mazzola;G. Carrea
焦碳酸二乙酯使四聚体3 α，20 β -羟基类固醇脱氢酶失活，在pH 6和25℃条件下，二级速率常数为1.63 M-1 s-1，在pH 9.4和25℃条件下，二级速率常数为190 M-1 s-1，羟胺作用后，活性缓慢而部分恢复（0.1 M羟胺，pH 9, 25℃条件下，28 h后活性恢复81%）。NADH保护酶的Kd值（10微米）非常接近辅酶的Km值（7微米）。灭活酶与天然酶的紫外光谱差异表明，在pH 6和25℃条件下，焦碳酸二乙酯修饰每个酶亚基的单个组氨酸残基，其二级速率常数为1.8 M-1 s-1。然而，组氨酸残基对活性不是必需的，因为在NADH存在的情况下，组氨酸残基的修饰没有酶失活，而失活酶的修饰被羟胺迅速逆转而没有伴随的再激活。在NAD+存在的情况下，黄体酮保护组氨酸残基不受修饰，这表明该残基位于酶的类固醇结合位点或附近。在处理过的酶上进行的二硝基苯基化实验证明，焦碳酸二乙酯也以异常高的反应速率修饰了每个酶亚基一个赖基残基。不同ph值赖氨酸残基的失活和修饰与NADH对赖氨酸残基失活和修饰的保护之间的相关性表明，该残基是酶活性所必需的，位于酶的NADH结合位点或附近。（摘要删节250字）
Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)