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 mechani sm by which this enzyme controls phosphatidylcholine synthesis, we have ini tiated studies of CCT from the model genetic system, the yeast Saccharomyce s cerevisiae. The yeast CCT gene was isolated from genomic DNA using the po lymerase chain reaction and was found to encode tyrosine at position 192 in stead of histidine, as originally reported. Levels of expression of yeast C CT activity in Escherichia coli or in the yeast, Pichia pastoris, were some what low. Expression of yeast CCT in a baculovirus system as a ex-His-tag f usion protein was higher and was used to purify yeast CCT by a procedure th at included delipidation, Kinetic characterization revealed that yeast CCT was activated approximately 20-fold by 20 muM phosphatidylcholine:oleate ve sicles, a level B-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 pho sphocholine did not change significantly upon activation by lipids; K-m val ues 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 phosphocholin e 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. (C) 2001 Academic P ress.