Mega T, Nishijima T, Ikenaka T
Department of Chemistry, Osaka University College of Science.
J Biochem. 1990 Apr;107(4):641-4. doi: 10.1093/oxfordjournals.jbchem.a123100.
beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.
β-D-吡喃半乳糖基甲基对硝基苯三氮烯（β-GalMNT）是一种β-半乳糖苷酶的特异性抑制剂，通过高效液相色谱法分离得到晶体，并对其化学和物理化学特性进行了研究。米曲霉β-半乳糖苷酶被该化合物灭活。我们详细研究了其抑制机制。该抑制剂被酶水解为对硝基苯胺和一种活性中间体（β-吡喃半乳糖基甲基碳正离子或β-吡喃半乳糖基甲基重氮鎓），该中间体使酶失活。酶的失活效率（失活酶的摩尔数与酶水解的β-GalMNT的摩尔数之比）为3%；大肠杆菌β-半乳糖苷酶的效率为49%。尽管效率较低，但米曲霉酶的失活速率与大肠杆菌酶的失活速率并没有太大差异，因为前者水解β-GalMNT的速度比后者快。米曲霉β-半乳糖苷酶也被β-吡喃半乳糖基甲基三氮烯的对氯苯基、对甲苯基和间硝基苯基衍生物灭活。然而，大肠杆菌β-半乳糖苷酶不会被这些三氮烯衍生物灭活。结果表明，β-GalMNT对米曲霉和大肠杆菌β-半乳糖苷酶的失活是一种酶激活的、活性位点导向的不可逆失活。对于大肠杆菌，排除了非酶促产生的中间体导致失活的可能性，但对于米曲霉酶则没有排除。
