Kanazawa H, Fujimoto S, Ohara A
Kyoto Pharmaceutical University, Japan.
Biol Pharm Bull. 1994 Jun;17(6):789-93. doi: 10.1248/bpb.17.789.
An inactivation mechanism of active papain (EC 3.4.22.2) by the Cu(2+)-ascorbic acid (AsA) system was examined. Incubation of active papain, which contains an active sulfhydryl (SH) group, with the Cu(2+)-AsA system under aerobic conditions resulted in an irreversible loss of enzyme activity. The enzyme was not inactivated at a molar ratio of enzyme to Cu2+ of 1:< 1, whereas at a molar ratio of 1:1-2, the extent of inactivation showed the same dependence on the extent of oxidation of AsA. Saturation kinetics were observed with respect to the concentration of AsA. The degree of inactivation was dependent on the decrease in SH content of the enzyme. Catalase at a low concentration partially protected the enzyme from inactivation, but did not affect the oxidation of AsA. In addition, catalase at a high concentration completely protected both the enzyme from inactivation and AsA from oxidation. The present results suggest that an additional function of H2O2, besides producing hydroxyl radicals (.OH), is to promote the conversion of Cu+ into Cu2+, and that an active SH group of papain is site-specifically modified by the .OH, resulting in inactivation of the enzyme.
研究了Cu(2+)-抗坏血酸（AsA）体系对活性木瓜蛋白酶（EC 3.4.22.2）的失活机制。在有氧条件下，将含有活性巯基（SH）的活性木瓜蛋白酶与Cu(2+)-AsA体系一起孵育，导致酶活性不可逆丧失。当酶与Cu2+的摩尔比为1:<1时，酶未失活，而当摩尔比为1:1-2时，失活程度与AsA的氧化程度呈现相同的依赖性。观察到AsA浓度的饱和动力学。失活程度取决于酶SH含量的降低。低浓度的过氧化氢酶部分保护酶不被失活，但不影响AsA的氧化。此外，高浓度的过氧化氢酶完全保护酶不被失活以及AsA不被氧化。目前的结果表明，除了产生羟基自由基（·OH）外，H2O2的另一个功能是促进Cu+转化为Cu2+，并且木瓜蛋白酶的活性SH基团被·OH位点特异性修饰，导致酶失活。
