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)
Yang
W L
WL
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903.
McDonough
V M
VM
Ozier-Kalogeropoulos
O
O
Adeline
M T
MT
Flocco
M T
MT
Carman
G M
GM
D001483:Base Sequence; D015153:Blotting, Western; D019731:Carbon-Nitrogen Ligases; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014544:Uridine Triphosphate / Q000378:metabolism
D011994:Recombinant Proteins; D005973:Glutamine; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D008274:Magnesium; D014544:Uridine Triphosphate