Bünning P, Kleemann S G, Riordan J F
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1990 Nov 20;29(46):10488-92. doi: 10.1021/bi00498a010.
The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-[3H]Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modified enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to greater than 95%) and the rest as N epsilon-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.
兔肺血管紧张素转换酶（ACE）的肽酶和酯酶活性在与1-氟-2,4-二硝基苯（Dnp-F）反应后迅速丧失。失活遵循试剂的一级动力学，并伴随着3,5-[³H]Dnp的化学计量掺入，表明该效应是由于酶的特异性修饰所致。对修饰酶的酸水解产物进行薄层色谱分析表明，大部分放射性标记以O-Dnp-酪氨酸（65%至大于95%）的形式存在，其余以Nε-Dnp-赖氨酸的形式存在。反应的pH依赖性与酪氨酸或赖氨酸的修饰一致。竞争性抑制剂的存在有效地保护酶不被Dnp-F失活。用N-乙酰咪唑对ACE进行乙酰化也能保护酶不被Dnp-F修饰。结果表明在ACE的活性位点存在催化必需的酪氨酸和赖氨酸残基。
