Legler G, Bieberich E
Institut für Biochemie, Universität Köln, Federal Republic of Germany.
Arch Biochem Biophys. 1988 Jan;260(1):437-42. doi: 10.1016/0003-9861(88)90467-5.
Hydrolysis of p-nitrophenyl-beta-D-glucoside by cytosolic beta-glucosidase proceeds with retention of the anomeric configuration. Whereas inactivation of the enzyme by the glucosidase inhibitor conduritol B epoxide (CBE) was extremely slow (ki(max)/Ki 0.57 M-1 min-1) it reacted 130 times more rapidly with 6-bromo-6-deoxy-CBE (Br-CBE). The beta-glucosidase could be labeled with [3H]Br-CBE; incorporation of 1 mol inhibitor/mol enzyme resulted in complete loss of activity. Most of the bound inhibitor was released after denaturation and treatment with ammonia as (1,3,4/2,5,6)-6-bromocyclohexanepentol, thus demonstrating the formation of an ester bond with an active site carboxylate by trans-diaxial opening of the epoxide ring. It was concluded from the Ki values for the epoxide inhibitors and for coduritol B with the cytosolic enzyme and corresponding data for the lysosomal beta-glucosidase that the unusually low reactivity with CBE and Br-CBE is probably due to the inability of the cytosolic enzyme to effectively donate a proton to the epoxide oxygen. An extremely rapid inactivation of the cytosolic beta-glucosidase was caused by bromoconduritol F ((1,2,4/3)-1-bromo-2,3,4-trihydroxycyclohex-5-ene) with ki(max)/Ki 10(5) M-1 min-1. In contrast with the Br-CBE-inhibited enzyme the beta-glucosidase inhibited by bromoconduritol F was subject to spontaneous reactivation with t1/2 approximately 20 min.
胞质β-葡萄糖苷酶催化对硝基苯基-β-D-葡萄糖苷水解时，端基异构构型得以保留。虽然葡萄糖苷酶抑制剂conduritol B环氧化物（CBE）使该酶失活的速度极慢（ki(max)/Ki为0.57 M-1 min-1），但它与6-溴-6-脱氧-CBE（Br-CBE）的反应速度要快130倍。β-葡萄糖苷酶可用[3H]Br-CBE进行标记；每摩尔酶掺入1摩尔抑制剂会导致活性完全丧失。大多数结合的抑制剂在变性并用氨处理后以(1,3,4/2,5,6)-6-溴环己五醇的形式释放出来，从而证明通过环氧化物环的反式双轴开环与活性位点羧酸盐形成了酯键。从环氧化物抑制剂和coduritol B对胞质酶的Ki值以及溶酶体β-葡萄糖苷酶的相应数据可以得出结论，与CBE和Br-CBE反应异常缓慢可能是由于胞质酶无法有效地向环氧化物氧提供质子。溴代conduritol F（(1,2,4/3)-1-溴-2,3,4-三羟基环己-5-烯）能使胞质β-葡萄糖苷酶极快速失活，ki(max)/Ki为10(5) M-1 min-1。与Br-CBE抑制的酶不同，溴代conduritol F抑制的β-葡萄糖苷酶会自发重新激活，半衰期约为20分钟。
