EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF CHOLINE KINASE, PRODUCT OF THE CKI GENE FROM SACCHAROMYCES-CEREVISIAE

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline pho sphotransferase, EC 2.7.1.32) is the product of the CKI gene, Choline kinase catalyzes the committed step in the synthesis of phosphatidylch oline by the CDP-choline pathway, The yeast enzyme was overexpressed 1 06-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity fro m the cytosolic fraction by chromatography with concanavalin A, Affi-G el Blue, and Mono Q, The N-terminal amino acid sequence of purified ch oline kinase matched perfectly with the deduced sequence of the CKI ge ne, The minimum subunit molecular mass (73 kDa) of purified choline ki nase was in good agreement with the predicted size (66.3 kDa) of the C RI gene product, Native choline kinase existed in oligomeric structure s of dimers, tetramers, and octomers, The amounts of the tetrameric an d octomeric forms increased in the presence of the substrate ATP, Anti bodies were raised against the purified enzyme and were used to identi fy choline kinase in insect cells and in S. cerevisiae, Maximum cholin e 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 indicate d that the reverse reaction was favored in vitro, The activation energ y for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respec t to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3), Results of the kinetic experiments indicated that the enzy me catalyzes a sequential Bi Bi reaction, The V-max for the reaction w as 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 kinas e subunit was 153 s(-1), Ethanolamine was a poor substrate for the pur ified 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 inhibitio n with respect to ATP and choline was complex. The regulation of choli ne kinase activity by ATP and ADP may be physiologically relevant.