Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by TC NMR spectroscopy

Journal Article · Mon Nov 25 04:00:00 EST 1985 · J. Biol. Chem.; (United States)

OSTI ID:6336275

Kohlbrenner, W E; Fesik, S W

(99%, 1- TC)- and (90%, 2- TC)3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by TC NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1- TC- or 2- TC-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the TC-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when (1- TC)- and (2- TC)KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP-KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-(1- TC)KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO.

• Research Organization: Abbott Labs., Abbott Park, IL
• OSTI ID: 6336275
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 27; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
CARBON 13
CARBON ISOTOPES
ENZYMES
ESCHERICHIA COLI
EVEN-ODD NUCLEI
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEOTIDYLTRANSFERASES
PHOSPHORUS-GROUP TRANSFERASES
RECEPTORS
RESONANCE
STABLE ISOTOPES
SUBSTRATES
TRANSFERASES