title: Purification, characterization, and high performance liquid chromatography assay of Salmonella glucose-1-phosphate cytidylyltransferase from the cloned rfbF gene.
abstract: We report the purification and characterization of glucose-1-phosphate cytidylyltransferase, the first of five enzymes committed to biosynthesis of CDP-D-abequose from Salmonella enterica strain LT2. The purification was greatly facilitated by using a cloned rfbF gene encoding this enzyme. Pure enzyme was obtained by 64-fold enrichment in three chromatography steps. The NH2-terminal sequence of the purified enzyme was in agreement with the sequence predicted from the nucleotide sequence of the rfbF gene. The SDS-polyacrylamide gel electrophoresis estimated subunit M(r) of 31,000 agrees well with the M(r) of 29,035 calculated from the amino acid composition deduced from the nucleotide sequence of the rfbF gene. The glucose-1-phosphate cytidylyltransferase catalyzes a reversible bimolecular group transfer reaction and steady-state kinetic measurements, including product inhibition patterns, indicate that this reaction proceeds by a "ping-pong" type of mechanism. The Km values for CTP, alpha-D-glucose 1-phosphate, CDP-D-glucose, and pyrophosphate are 0.28, 0.64, 0.11, and 1.89 mM, respectively.
authors.affiliation: Department of Clinical Bacteriology, Karolinska Institute, Huddinge Hospital, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Lindqvist
authors.forename: R
authors.initials: R
authors.lastname: Kaiser
authors.forename: P R
authors.initials: PR
authors.lastname: Reeves
authors.forename: A A
authors.initials: AA
authors.lastname: Lindberg
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012475:Salmonella / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D009713:Nucleotidyltransferases; C020586:glucose-1-phosphate cytidylyltransferase