title: 13C NMR investigation of the anomeric specificity of CMP-N-acetylneuraminic acid synthetase from Escherichia coli.
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.
authors.affiliation: Laboratory of Bacterial Polysaccharides, Food and Drug Administration, Bethesda, Maryland 20892.
authors.forename: M G
authors.initials: MG
authors.lastname: Ambrose
authors.forename: S J
authors.initials: SJ
authors.lastname: Freese
authors.forename: M S
authors.initials: MS
authors.lastname: Reinhold
authors.forename: T G
authors.initials: TG
authors.lastname: Warner
authors.forename: W F
authors.initials: WF
authors.lastname: Vann
mesh_terms: D002236:Carbohydrate Conformation; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology*; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D015394:Molecular Structure; D000216:N-Acylneuraminate Cytidylyltransferase / Q000378:metabolism*; D012794:Sialic Acids / Q000138:chemical synthesis* / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D002247:Carbon Isotopes; D007202:Indicators and Reagents; D012794:Sialic Acids; D000216:N-Acylneuraminate Cytidylyltransferase