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Biochemistry. 1992 Jan 28;31(3):775-80.

13C NMR investigation of the anomeric specificity of CMP-N-acetylneuraminic acid synthetase from Escherichia coli.

Author information

1
Laboratory of Bacterial Polysaccharides, Food and Drug Administration, Bethesda, Maryland 20892.

Abstract

The anomeric specificity of Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was investigated by NMR using 13C-labeled N-acetylneuraminic acid (NeuAc). Consumption of the beta-anomer of [2-13C]N-acetylneuraminic acid was observed upon addition of enzyme, with a concomitant appearance of an anomeric resonance for CMP-N-acetylneuraminic acid. Inhibition by substrate analogues the anomeric oxygen was determined in a similar manner using [2-13C,(50 atom %)18O]N-acetylneuraminic acid. An upfield shift of 1.5 Hz in the anomeric resonance of both the [13C]NeuAc substrate and CMP-[13C]NeuAc product was observed due to the 18O substitution. This result implies conservation of the NeuAc oxygen. Results of steady-state kinetic analysis suggest a sequential-type mechanism and therefore no covalent intermediate. Thus, CMP-beta-NeuAc is probably formed by a direct transfer of the anomeric oxygen of beta-NeuAc to the alpha-phosphate of CTP.

PMID:
1731934
DOI:
10.1021/bi00118a019
[Indexed for MEDLINE]

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