The inactivation of glucosamine synthetase from bacteria by anticapsin, the C-terminal epoxyamino acid of the antibiotic tetaine.
Incubation of anticapsin with the purified glucosamine synthetase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase, amino transferring, EC 5.3.1.19) from Escherichia coli, Pseudomonas aeruginosa, Arthrobacter aurescens and Bacillus thuringiensis led to the formation of an inactive enzyme irreversibly modified. The inactivation reaction followed pseudo-first-order kinetics. The rate of the inactivation reaction at various concentrations of anticapsin exhibited saturation kinetics, implying that anticapsin binds reversibly to the enzyme prior to inactivation. The determined Kinact is in the range of 10(-5) M (B. thuringiensis) and 10(-6) M (E. coli, P. aeruginosa, A. aurescens ). The addition of glutamine protected the amidotransferase from inactivation by anticapsin . The anticapsin was demonstrated to be a mixed type or competitive inhibitor with respect to glutamine with a Ki value of 10(-6) to 10(-7) M. Reaction of anticapsin with the enzyme exhibits the characteristics of affinity labelling of the glutamine binding site. Chemical modification of the enzyme thiol group with various reagents, 5,5'-dithiobis-(2-nitrobenzoic) acid, 6,6'- dithiodinicotinic acid, 1,1'- dithiodiformamidine , N-ethylmaleimide and iodoacetamide, resulted in an inactive enzyme.
D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001173:Arthrobacter / Q000201:enzymology*; D001413:Bacillus thuringiensis / Q000201:enzymology*; D001667:Binding, Competitive; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000031:analogs & derivatives / Q000494:pharmacology; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors*; D007700:Kinetics; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D005973:Glutamine; C000097:anticapsin; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D002238:Carbohydrate Epimerases; D000409:Alanine