Purification and Characterization of CTP Synthetase, the Product of the URA7 Gene in Saccharomyces cerevisiae

Abstract

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)

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

“…The CTP product provides a negative feedback by competitively inhibiting the UTP substrate [10,15,[20][21][22][23]. Despite bacterial and mammalian lineages having diverged approximately three billion years ago [24], their CTP synthase molecules Filament-formation appears to be a conserved, and thus an essential, property of CTP synthases.…”

Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces cerevisiae

“…GTP stimulates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (2)(3)(4)(5). URA7 (6) and URA8 (7) are duplicate genes that code for CTP synthetase in S. cerevisiae.…”

“…CTP synthetase activity is allosterically regulated by its substrates and product CTP. The enzyme exhibits positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (5,28). The positive cooperative kinetics of the URA7-encoded enzyme with respect to UTP and ATP are due to the nucleotide-dependent oligomerization of an inactive dimeric form to an active tetrameric form of the enzyme (29).…”

“…The positive cooperative kinetics of the URA7-encoded enzyme with respect to UTP and ATP are due to the nucleotide-dependent oligomerization of an inactive dimeric form to an active tetrameric form of the enzyme (29). A major form of CTP synthetase regulation is mediated by CTP product inhibition (5,28). CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP (5).…”

“…A major form of CTP synthetase regulation is mediated by CTP product inhibition (5,28). CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP (5). Amino acid residue Glu 161 has been identified as being involved in this regulation (see Fig.…”