Purification and kinetic characterization of CTP:phosphocholine cytidylyltransferase from Saccharomyces cerevisiae.
CTP:phosphocholine cytidylyltransferase (CCT) regulates the biosynthesis of phosphatidylcholine in mammalian cells. In order to understand the mechanism by which this enzyme controls phosphatidylcholine synthesis, we have initiated studies of CCT from the model genetic system, the yeast Saccharomyces cerevisiae. The yeast CCT gene was isolated from genomic DNA using the polymerase chain reaction and was found to encode tyrosine at position 192 instead of histidine, as originally reported. Levels of expression of yeast CCT activity in Escherichia coli or in the yeast, Pichia pastoris, were somewhat low. Expression of yeast CCT in a baculovirus system as a 6x-His-tag fusion protein was higher and was used to purify yeast CCT by a procedure that included delipidation. Kinetic characterization revealed that yeast CCT was activated approximately 20-fold by 20 microM phosphatidylcholine:oleate vesicles, a level 5-fold lower than that necessary for maximal activation of rat CCT. The k(cat) value was 31.3 s(-1) in the presence of lipid and 1.5 s(-1) in the absence of lipid. The K(m) values for the substrates CTP and phosphocholine did not change significantly upon activation by lipids; K(m) values in the presence of lipid were 0.80 mM for phosphocholine and 1.4 mM for CTP while K(m) values in the absence of lipid were 1.2 mM for phosphocholine and 0.8 mM for CTP. Activation of yeast CCT, therefore, appears to be due to an increase in the k(cat) value upon lipid binding.
Friesen
J A
JA
Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, MI 48109-0606, USA.
Park
Y S
YS
Kent
C
C
D000595:Amino Acid Sequence; D000906:Antibodies; D015153:Blotting, Western; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000276:immunology; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*
D000906:Antibodies; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019916:Choline-Phosphate Cytidylyltransferase