Shapiro R, Riordan J F
Biochemistry. 1983 Nov 8;22(23):5315-21. doi: 10.1021/bi00292a010.
Pulmonary angiotensin converting enzyme has been reductively methylated by using formaldehyde and sodium cyanoborohydride. This modification virtually eliminates enzyme activity toward some substrates (e.g., furanacryloyl-Phe-Gly-Gly) while less drastically affecting activity toward others (e.g., furanacryloyl-Phe-Phe-Arg). Affinity chromatography and analysis of radiolabeled reaction products reveal that this effect is due to methylation of a single critical lysine residue. Loss of activity primarily represents an increase in Km values, indicating that the critical lysine plays a role in substrate binding. This lysine can be protected by a competitive inhibitor, suggesting that it is at or near the active site. Addition of chloride at pH 6.1 specifically protects against methylation of this lysine. These findings support the idea that the critical lysine is part of the binding site for chloride and other monovalent anions which are strong activators of the enzyme.
利用甲醛和氰基硼氢化钠对肺血管紧张素转换酶进行了还原甲基化修饰。这种修饰几乎消除了该酶对某些底物（如呋喃丙烯酰 - 苯丙氨酸 - 甘氨酸 - 甘氨酸）的活性，而对其他底物（如呋喃丙烯酰 - 苯丙氨酸 - 苯丙氨酸 - 精氨酸）活性的影响则较小。亲和层析及对放射性标记反应产物的分析表明，这种效应是由于单个关键赖氨酸残基的甲基化所致。活性丧失主要表现为米氏常数（Km）值的增加，这表明关键赖氨酸在底物结合中起作用。该赖氨酸可被竞争性抑制剂保护，提示它位于活性位点或其附近。在pH 6.1时添加氯离子可特异性地防止该赖氨酸的甲基化。这些发现支持了这样一种观点，即关键赖氨酸是氯离子及其他一价阴离子结合位点的一部分，而这些离子是该酶的强激活剂。
