Chemical modification of glucosamine-6-phosphate synthase by diethyl pyrocarbonate: evidence of histidine requirement for enzymatic activity. |
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Authors: | M A Badet-Denisot B Badet |
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Affiliation: | Laboratoire de Bioorganique & Biotechnologies, UA CNRS 1389 ENSCP, Paris, France. |
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Abstract: | 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. |
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