CTP synthetase from Escherichia coli: an improved purification procedure and characterization of hysteretic and enzyme concentration effects on kinetic properties

Abstract

Previous studies have shown that CTP synthetase exists as a dimer which aggregates to a tetramer in the presence of the substrates ATP and UTP [Long, C. W., Levitzki, A., & Koshland, D. E., Jr. (1970) J. Biol. Chem. 245, 80]. A new, relatively simple purification procedure resulting in enzyme of high purity and in good yield has been established by using two successive hydrophobic chromatography steps, the first in the absence of ATP and UTP (dimer binds) and the second in the presence of ATP and UTP (tetramer does not bind). Several previously unreported properties of CTP synthetase are described which suggest that alterations in the state of association and dissociation of the enzyme have a controlling influence on the observed kinetic properties of the enzyme. The specific activity of CTP synthetase decreases with decreasing enzyme concentration, particularly when the concentrations of ATP and UTP in the reaction mixture are nonsaturating. The concentration of ATP or UTP required for half-maximal activity is significantly increased as the concentration of enzyme in the reaction mixture is decreased. CTP synthetase displays reversible cold lability and hysteretic properties (lags or bursts in the time course of product formation), both of which are influenced by the concentration of enzyme and/or the presence of ATP and UTP in the preincubation mixture and/or assay mixture. Gel filtration studies have shown that CTP synthetase can dissociate to an apparently inactive monomer. The dissociation is reversible, and the rate of association is slow.

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex,

“…The extensive interdependent contacts, particularly with the γ-phosphate, provide an explanation for the absolute requirement for triphosphate substrate and regulator, as well as the requirement for conversion of noninhibitory CPEC and 3deazaU in vivo to the inhibitory nucleoside triphosphates (22,50,58). The thermodynamic linkage between substrate binding and the assembly of competent CTP synthesis sites clearly explains the apparent positive cooperativity of ATP and UTP at subsaturating substrate concentrations, and supports the idea that CTPSs are regulated in part by an associationdissociation mechanism (3,11,12). In addition, the product-inhibited enzyme is still in the "active" tetrameric form, and is conceivably poised to receive substrates, potentially allowing for a faster response to changing metabolic needs for CTP.…”

“…In addition, the product-inhibited enzyme is still in the "active" tetrameric form, and is conceivably poised to receive substrates, potentially allowing for a faster response to changing metabolic needs for CTP. Indeed, at physiological enzyme concentrations, a significant lag in activity is observed if EcCTPS is not preincubated with UTP or ATP (12), and at low enzyme concentrations, CTP stimulates activity, presumably by promoting tetramerization (3,13). The ability to exchange the pyrimidine ring between sites without shifting the triphosphate suggests the possibility that CTP might bind the inhibitory site immediately after synthesis and prior to dissociating from the enzyme.…”

“…1 Abbreviations: CTPS, cytidine triphosphate synthetase; CPEC, cyclopentenylcytosine; 3deazaU, 3-deazauridine; EcCTPS, Escherichia coli cytidine triphosphate synthetase; ALase, ammonia ligase reaction, i.e., the formation of a carbon-nitrogen bond by displacement of a phosphorylated oxygen; GATase, glutamine amidotransferase, which refers to a conserved glutamine hydrolysis domain that provides ammonia for a number of diverse enzyme-catalyzed reactions; DTBS, E. coli dethiobiotin synthetase (BioD) enzyme; rmsd, root-meansquare difference in position between atom sets, in angstroms. (Figure 1b) (3,4,(11)(12)(13). Further, the product CTP provides negative feedback by acting as a competitive inhibitor of substrate UTP (3,8,9,14), with a comparable K i for CTP (110 μM) and K m for UTP (150 μM) (3) (Figure 1a).…”

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

“…Both adenosine-5 0 -triphosphate (ATP) and uridine-5 0 -triphosphate (UTP), as substrates, bind CTP synthase with positive co-operativity [10][11][12][13][14][15][16].…”

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

“…CTP synthetase enzymes have been isolated from both prokaryotic and eukaryotic organisms [2,21,22,[24][25][26][27]. Moreover, structures for the bacterial [28,29] and human [30] enzymes have been solved.…”