title: Inhibition of Escherichia coli CTP Synthetase by NADH and Other Nicotinamides and Their Mutual Interactions with CTP and GTP.
abstract: CTP synthetases catalyze the last step of pyrimidine biosynthesis and provide the sole de novo source of cytosine-containing nucleotides. As a central regulatory hub, they are regulated by ribonucleotide and enzyme concentration through ATP and UTP substrate availability, CTP product inhibition, GTP allosteric modification, and quaternary structural changes including the formation of CTP-inhibited linear polymers (filaments). Here, we demonstrate that nicotinamide redox cofactors are moderate inhibitors of Escherichia coli CTP synthetase (EcCTPS). NADH and NADPH are the most potent, and the primary inhibitory determinant is the reduced nicotinamide ring. Although nicotinamide inhibition is noncompetitive with substrates, it apparently enhances CTP product feedback inhibition and GTP allosteric regulation. Further, CTP and GTP also enhance each other's effects, consistent with the idea that NADH, CTP, and GTP interact with a common intermediate enzyme state. A filament-blocking mutation that reduces CTP inhibitory effects also reduced inhibition by GTP but not NADH. Protein-concentration effects on GTP inhibition suggest that, like CTP, GTP preferentially binds to the filament. All three compounds display nearly linear dose-dependent inhibition, indicating a complex pattern of cooperative interactions between binding sites. The apparent synergy between inhibitors, in consideration with physiological nucleotide concentrations, points to metabolically relevant inhibition by nicotinamides, and implicates cellular redox state as a regulator of pyrimidine biosynthesis.
authors: Department of Molecular and Cellular Biology, University of California, Davis , Davis, California 95616, United States.|Biophysics Graduate Program, University of California, Berkeley , Berkeley, California 94720, United States.
authors: Chris
authors: C
authors: Habrian
authors: Department of Molecular and Cellular Biology, University of California, Davis , Davis, California 95616, United States.|Process Development Rotation Program, Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
authors: Adithi
authors: A
authors: Chandrasekhara
authors: Department of Molecular and Cellular Biology, University of California, Davis , Davis, California 95616, United States.
authors: Bita
authors: B
authors: Shahrvini
authors: Department of Molecular and Cellular Biology, University of California, Davis , Davis, California 95616, United States.|Department of Biology, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors: Brian
authors: B
authors: Hua
authors: Department of Molecular and Cellular Biology, University of California, Davis , Davis, California 95616, United States.|Drexel University College of Medicine , Philadelphia, Pennsylvania 19129, United States.|Kaiser Permanente , Sacramento, California 95823, United States.
authors: Jason
authors: J
authors: Lee
authors: Department of Molecular and Cellular Biology, University of California, Davis , Davis, California 95616, United States.
authors: Roger
authors: R
authors: Jesinghaus
authors: Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, United States.|Department of Cellular and Molecular Medicine, University of California, San Diego , La Jolla, California 92903, United States.
authors: Rachael
authors: R
authors: Barry
authors: Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, United States.
authors: Zemer
authors: Z
authors: Gitai
authors: Department of Biochemistry, University of Washington , Seattle, Washington 98195, United States.
authors: Justin
authors: J
authors: Kollman
authors: Department of Molecular and Cellular Biology, University of California, Davis , Davis, California 95616, United States.
authors: Enoch P
authors: EP
authors: Baldwin
mesh_terms: D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism*
chemical_list: D009243:NAD; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase