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 CKI 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 Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, 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.

Purification and characterization of CTP synthetase, the product of the URA7 gene in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, CTP synthetase [EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] is the product of the URA7 gene. CTP synthetase was purified 503-fold to apparent homogeneity from cells bearing the URA7 gene on a multicopy plasmid that directed a 10-fold overproduction of the enzyme. The purification procedure included ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Sephacryl 300 HR, Q-Sepharose, Affi-Gel Blue, and Superose 6. The N-terminal amino acid sequence of purified CTP synthetase was identified and aligned perfectly with the deduced sequence of the URA7 gene. The minimum subunit molecular mass (68 kDa) of purified CTP synthetase was in good agreement with the size (64.7 kDa) of the URA7 gene product. Antibodies were raised against a maltose-binding protein-CTP synthetase fusion protein which immunoprecipitated CTP synthetase from wild-type cells. Immunoblot analysis was used to identify CTP synthetase in wild-type cells and cells bearing the URA7 gene on a multicopy plasmid. The results of gel filtration chromatography indicated that the size of native CTP synthetase was consistent with a dimeric structure for the enzyme. CTP synthetase oligomerized to a tetramer in the presence of its substrates UTP and ATP. Maximum CTP synthetase activity was dependent on magnesium ions (4 mM) and 2-mercaptoethanol at the pH optimum of 8.0. CTP synthetase exhibited positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP. CTP synthetase was potently inhibited by the product CTP which also increased the positive cooperativity of the enzyme toward UTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide-binding specificity of the molecular chaperone BiP.

Members of the heat-shock protein family (hsp70s) can distinguish folded from unfolded proteins. This property is crucial to the role of hsp70s as molecular chaperones and is attributable to the amino-acid specificity of the peptide-binding site. The specificity for peptide ligands is investigated using a set of peptides of random sequence but defined chain length. The peptide-binding site selects for aliphatic residues and accommodates them in an environment energetically equivalent to the interior of a folded protein.