Nakanishi Y, Isohashi F, Matsunaga T, Sakamoto Y
Eur J Biochem. 1985 Oct 15;152(2):337-42. doi: 10.1111/j.1432-1033.1985.tb09203.x.
The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition of AsA plus Cu2+ was mainly due to attack on its active site.
从大鼠肝脏上清液部分纯化得到的乙酰辅酶A水解酶（二聚体形式）的活性在0℃时半衰期（t1/2）为3分钟，但在37℃时稳定（t1/2 = 34小时）[Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584 - 590]。在37℃下将纯化的酶与L - 抗坏血酸（AsA）一起孵育会导致酶失活（在2 mM AsA时t1/2 = 90分钟）。添加过渡金属离子（Cu2 +、Fe2 +和Fe3 +）会大大增强失活程度。硫醇还原剂，如还原型谷胱甘肽和DL - 二硫苏糖醇，可保护水解酶不被AsA失活。然而，这些物质不能恢复被AsA失活的酶的催化活性。当含有Cu2 +的AsA溶液在有氧条件下于37℃预孵育不同时间（无酶），然后取等分试样与酶溶液孵育20分钟时，发现剩余活性随预孵育时间增加而降低，在60分钟时达到最低。然而，进一步预孵育会降低失活的可能性。过氧化氢酶，一种过氧化氢（H2O2）清除剂，几乎完全阻止了AsA加Cu2 +对酶的失活作用。超氧化物歧化酶和钛铁试剂，两者都是超氧阴离子（O2 - ）清除剂，也阻止了酶的失活。高浓度的甘露醇，一种羟基自由基（OH）清除剂，部分保护酶不被失活。这些结果表明，在Cu2 +存在下AsA对酶的失活是由于活性氧物种（H2O2、O2 - 、OH）的作用，这些活性氧物种是已知的AsA自氧化产物。戊酰辅酶A，乙酰辅酶A水解酶的竞争性抑制剂，极大地保护酶不被AsA加Cu2 +失活，但ATP和ADP这两种该酶的效应物只有轻微的保护作用。这些结果表明，添加AsA加Cu2 +对该酶的失活主要是由于对其活性位点的攻击。
