title: Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.
abstract: 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.
authors.affiliation: Department of Chemistry, Osaka University College of Science.
authors.forename: T
authors.initials: T
authors.lastname: Mega
authors.forename: T
authors.initials: T
authors.lastname: Nishijima
authors.forename: T
authors.initials: T
authors.lastname: Ikenaka
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D001236:Aspergillus oryzae / Q000187:drug effects / Q000201:enzymology*; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D005696:Galactosidases / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D014226:Triazenes / Q000378:metabolism*; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors*
chemical_list: D014226:Triazenes; C018547:beta-D-galactopyranosylmethyl-4-nitrophenyltriazene; D005696:Galactosidases; D001616:beta-Galactosidase