Differential biochemical regulation of the URA7- and URA8-encoded CTP synthetases from Saccharomyces cerevisiae.
The URA7- and URA8-encoded CTP synthetases (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming) are functionally overlapping enzymes responsible for the biosynthesis of CTP in the yeast Saccharomyces cerevisiae. URA8-encoded CTP synthetase was purified to apparent homogeneity by ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Q-Sepharose, Affi-Gel Blue, Mono Q, and Superose 6. The subunit molecular mass (67 kDa) of purified URA8-encoded CTP synthetase was in good agreement with the predicted size of the URA8 gene product. Antibodies raised against a fusion protein constructed from the coding sequences of the URA8 gene and expressed in Escherichia coli reacted with purified URA8-encoded CTP synthetase. Native URA8-encoded CTP synthetase existed as a dimer which oligomerized to a tetramer in the presence of its substrates UTP and ATP. Maximum URA8-encoded CTP synthetase activity was dependent on Mg2+ ions (Ka = 2.4 mM) and 2-mercaptoethanol at the pH optimum of 7.5. The enzyme followed saturation kinetics toward UTP (Km = 74 microM), ATP (Km = 22 microM), and glutamine (Km = 0.14 mM). GTP stimulated (Ka = 26 microM) URA8-encoded CTP synthetase activity 12-fold. CTP potently inhibited (IC50 = 85 microM) URA8-encoded CTP synthetase activity and, in addition, caused the dependence of activity toward UTP to become cooperative. The URA8-encoded CTP synthetase and the previously purified URA7-encoded CTP synthetase differed significantly with respect to several biochemical properties including turnover number, pH optimum, substrate dependences, and sensitivity to inhibition by CTP. The URA7-encoded CTP synthetase mRNA was 2-fold more abundant when compared with URA8-encoded CTP synthetase mRNA. Both CTP synthetase isoforms were maximally expressed in the exponential phase of growth.
Nadkarni
A K
AK
Department of Food Science, Cook College, Rutgers University, New Brunswick, New Jersey 08903, USA.
McDonough
V M
VM
Yang
W L
WL
Stukey
J E
JE
Ozier-Kalogeropoulos
O
O
Carman
G M
GM
D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases; D003570:Cytidine Triphosphate / Q000494:pharmacology; D005800:Genes, Fungal; D008025:Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D012333:RNA, Messenger; D003570:Cytidine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase