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

Journal Article · Tue Jan 28 04:00:00 EST 1992 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00118a019· OSTI ID:5488926

Ambrose, M G; Freese, S J; Reinhold, M S; Vann, W F ^[1]; Warner, T G ^[2]

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1. Food and Drug Administration, Bethesda, MD (United States)
2. Genentech Inc., San Francisco, CA (United States)

The anomeric specificity of Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was investigated by NMR using {sup 13}C-labeled N-acetylneuraminic acid (NeuAc). Consumption of the {beta}-anomer of (2-{sup 13}C)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 confirms the importance of the anomeric center for interaction of substrate with the enzyme. The fate of the anomeric oxygen was determined in a similar manner using (2-{sup 13}C,(50 atom %){sup 18}O)N-acetylneuraminic acid. An upfield shift of 1.5 Hz in the anomeric resonance of both the ({sup 13}C)NeuAc substrate and CMP-({sup 13}C)NeuAc product was observed due to the {sup 18}O 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.

• OSTI ID: 5488926
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 31:3; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BARYONS
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
FERMIONS
HADRONS
ISOTOPES
KETO ACIDS
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NITROGEN TRANSFERASES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXYGEN 18
OXYGEN ISOTOPES
POLYSACCHARIDES
PROTEINS
PROTONS
PYRUVIC ACID
REACTION KINETICS
RESONANCE
SACCHARIDES
STABLE ISOTOPES
TRANSFERASES