Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene, Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway, The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q, The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene, The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CRI gene product, Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers, The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP, Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae, Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C, The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro, The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3), Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction, The V-max for the reaction was 138.7 mu mol/min/mg, and the K-m values for choline and ATP were 0.27 mM and 90 mu M, respectively, The turnover number per choline kinase subunit was 153 s(-1), Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity, ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.