Chemical modification of glucosamine-6-phosphate synthase by diethyl pyrocarbonate: evidence of histidine requirement for enzymatic activity.
Glucosamine-6-phosphate synthase from Escherichia coli was inactivated by diethylpyrocarbonate at pH 7.3 and 4 degrees C with a second-order rate constant of 1220 M-1 min-1. The difference spectrum of inactivated vs native enzyme had a maximum absorption at 242 nm, which is characteristic of N-carbethoxyhistidine. No trough at around 280 nm due to O-carbethoxytyrosine was observed and the sulfhydryl content of the enzyme was unchanged. Studies with [14C]diethylpyrocarbonate provided evidence that derivatization of a single histidine residue of the amino-terminal glutamine-binding domain inactivated glucosamine-6P synthase. These results are consistent with the participation of an histidine residue in a catalytic triad, Cys/His/Asp, necessary to generate ammonia from glutamine.
Badet-Denisot
M A
MA
Laboratoire de Bioorganique & Biotechnologies, UA CNRS 1389 ENSCP, Paris, France.
Badet
B
B
D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis
D013438:Sulfhydryl Compounds; D006639:Histidine; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D004047:Diethyl Pyrocarbonate