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. 2000 Jan;9(1):53–63. doi: 10.1110/ps.9.1.53

The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase.

J B Sakash 1, A Tsen 1, E R Kantrowitz 1
PMCID: PMC2144450  PMID: 10739247

Abstract

As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5'-triphosphate (PTP), 6-chloropurine 5'-triphosphate (Cl-PTP), 6-mercaptopurine 5'-triphosphate (SH-PTP), 6-methylpurine 5'-triphosphate (Me-PTP), and 1-methyladenosine 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of L-aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2'-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2-carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect.

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Selected References

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  1. Banerjee A., Levy H. R., Levy G. C., Chan W. W. Conformations of bound nucleoside triphosphate effectors in aspartate transcarbamylase. Evidence for the London-Schmidt model by transferred nuclear Overhauser effects. Biochemistry. 1985 Mar 26;24(7):1593–1598. doi: 10.1021/bi00328a003. [DOI] [PubMed] [Google Scholar]
  2. Changeux J. P., Gerhart J. C., Schachman H. K. Allosteric interactions in aspartate transcarbamylase. I. Binding of specific ligands to the native enzyme and its isolated subunits. Biochemistry. 1968 Feb;7(2):531–538. doi: 10.1021/bi00842a007. [DOI] [PubMed] [Google Scholar]
  3. Corder T. S., Wild J. R. Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site. J Biol Chem. 1989 May 5;264(13):7425–7430. [PubMed] [Google Scholar]
  4. Dutta M., Kantrowitz E. R. The influence of the regulatory chain amino acids Glu-62 and IIe-12 on the heterotropic properties of Escherichia coli aspartate transcarbamoylase. Biochemistry. 1998 Jun 16;37(24):8653–8658. doi: 10.1021/bi980456h. [DOI] [PubMed] [Google Scholar]
  5. Evans S. V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graph. 1993 Jun;11(2):134-8, 127-8. doi: 10.1016/0263-7855(93)87009-t. [DOI] [PubMed] [Google Scholar]
  6. GERHART J. C., PARDEE A. B. The enzymology of control by feedback inhibition. J Biol Chem. 1962 Mar;237:891–896. [PubMed] [Google Scholar]
  7. Gerhart J. C., Holoubek H. The purification of aspartate transcarbamylase of Escherichia coli and separation of its protein subunits. J Biol Chem. 1967 Jun 25;242(12):2886–2892. [PubMed] [Google Scholar]
  8. Gouaux J. E., Stevens R. C., Lipscomb W. N. Crystal structures of aspartate carbamoyltransferase ligated with phosphonoacetamide, malonate, and CTP or ATP at 2.8-A resolution and neutral pH. Biochemistry. 1990 Aug 21;29(33):7702–7715. doi: 10.1021/bi00485a020. [DOI] [PubMed] [Google Scholar]
  9. Gray C. W., Chamberlin M. J., Gray D. M. Interaction of aspartate transcarbamylase with regulatory nucleotides. J Biol Chem. 1973 Sep 10;248(17):6071–6079. [PubMed] [Google Scholar]
  10. Honzatko R. B., Lipscomb W. N. Interactions of metal-nucleotide complexes with aspartate carbamoyltransferase in the crystalline state. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7171–7174. doi: 10.1073/pnas.79.23.7171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Honzatko R. B., Lipscomb W. N. Interactions of phosphate ligands with Escherichia coli aspartate carbamoyltransferase in the crystalline state. J Mol Biol. 1982 Sep 15;160(2):265–286. doi: 10.1016/0022-2836(82)90176-0. [DOI] [PubMed] [Google Scholar]
  12. Kantrowitz E. R., Lipscomb W. N. Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition. Trends Biochem Sci. 1990 Feb;15(2):53–59. doi: 10.1016/0968-0004(90)90176-c. [DOI] [PubMed] [Google Scholar]
  13. Kosman R. P., Gouaux J. E., Lipscomb W. N. Crystal structure of CTP-ligated T state aspartate transcarbamoylase at 2.5 A resolution: implications for ATCase mutants and the mechanism of negative cooperativity. Proteins. 1993 Feb;15(2):147–176. doi: 10.1002/prot.340150206. [DOI] [PubMed] [Google Scholar]
  14. Krause K. L., Volz K. W., Lipscomb W. N. 2.5 A structure of aspartate carbamoyltransferase complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate. J Mol Biol. 1987 Feb 5;193(3):527–553. doi: 10.1016/0022-2836(87)90265-8. [DOI] [PubMed] [Google Scholar]
  15. Ladjimi M. M., Ghellis C., Feller A., Cunin R., Glansdorff N., Piérard A., Hervé G. Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. II. Involvement of the C-terminal region of the regulatory chain in homotropic and heterotropic interactions. J Mol Biol. 1985 Dec 20;186(4):715–724. doi: 10.1016/0022-2836(85)90391-2. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lipscomb W. N. Aspartate transcarbamylase from Escherichia coli: activity and regulation. Adv Enzymol Relat Areas Mol Biol. 1994;68:67–151. doi: 10.1002/9780470123140.ch3. [DOI] [PubMed] [Google Scholar]
  18. London R. E., Schmidt P. G. A model for nucleotide regulation of aspartate transcarbamylase. Biochemistry. 1972 Aug 1;11(16):3136–3142. doi: 10.1021/bi00766a029. [DOI] [PubMed] [Google Scholar]
  19. MONOD J., WYMAN J., CHANGEUX J. P. ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. J Mol Biol. 1965 May;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
  20. Matsumoto S., Hammes G. G. An equilibrium binding study of the interaction of aspartate transcarbamylase with cytidine 5'-triphosphate and adenosine 5'-triphosphate. Biochemistry. 1973 Mar 27;12(7):1388–1394. doi: 10.1021/bi00731a019. [DOI] [PubMed] [Google Scholar]
  21. Monaco H. L., Crawford J. L., Lipscomb W. N. Three-dimensional structures of aspartate carbamoyltransferase from Escherichia coli and of its complex with cytidine triphosphate. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5276–5280. doi: 10.1073/pnas.75.11.5276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  23. Nowlan S. F., Kantrowitz E. R. Superproduction and rapid purification of Escherichia coli aspartate transcarbamylase and its catalytic subunit under extreme derepression of the pyrimidine pathway. J Biol Chem. 1985 Nov 25;260(27):14712–14716. [PubMed] [Google Scholar]
  24. ORNSTEIN L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964 Dec 28;121:321–349. doi: 10.1111/j.1749-6632.1964.tb14207.x. [DOI] [PubMed] [Google Scholar]
  25. Pastra-Landis S. C., Foote J., Kantrowitz E. R. An improved colorimetric assay for aspartate and ornithine transcarbamylases. Anal Biochem. 1981 Dec;118(2):358–363. doi: 10.1016/0003-2697(81)90594-7. [DOI] [PubMed] [Google Scholar]
  26. Robey E. A., Schachman H. K. Regeneration of active enzyme by formation of hybrids from inactive derivatives: implications for active sites shared between polypeptide chains of aspartate transcarbamoylase. Proc Natl Acad Sci U S A. 1985 Jan;82(2):361–365. doi: 10.1073/pnas.82.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sakash J. B., Kantrowitz E. R. The N-terminus of the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for both nucleotide binding and heterotropic effects. Biochemistry. 1998 Jan 6;37(1):281–288. doi: 10.1021/bi972102g. [DOI] [PubMed] [Google Scholar]
  28. Smith K. A., Nowlan S. F., Middleton S. A., O'Donovan C., Kantrowitz E. R. Involvement of tryptophan 209 in the allosteric interactions of Escherichia coli aspartate transcarbamylase using single amino acid substitution mutants. J Mol Biol. 1986 May 5;189(1):227–238. doi: 10.1016/0022-2836(86)90393-1. [DOI] [PubMed] [Google Scholar]
  29. Stebbins J. W., Xu W., Kantrowitz E. R. Three residues involved in binding and catalysis in the carbamyl phosphate binding site of Escherichia coli aspartate transcarbamylase. Biochemistry. 1989 Mar 21;28(6):2592–2600. doi: 10.1021/bi00432a037. [DOI] [PubMed] [Google Scholar]
  30. Stevens R. C., Chook Y. M., Cho C. Y., Lipscomb W. N., Kantrowitz E. R. Escherichia coli aspartate carbamoyltransferase: the probing of crystal structure analysis via site-specific mutagenesis. Protein Eng. 1991 Apr;4(4):391–408. doi: 10.1093/protein/4.4.391. [DOI] [PubMed] [Google Scholar]
  31. Stevens R. C., Gouaux J. E., Lipscomb W. N. Structural consequences of effector binding to the T state of aspartate carbamoyltransferase: crystal structures of the unligated and ATP- and CTP-complexed enzymes at 2.6-A resolution. Biochemistry. 1990 Aug 21;29(33):7691–7701. doi: 10.1021/bi00485a019. [DOI] [PubMed] [Google Scholar]
  32. Stevens R. C., Lipscomb W. N. Allosteric control of quaternary states in E. coli aspartate transcarbamylase. Biochem Biophys Res Commun. 1990 Sep 28;171(3):1312–1318. doi: 10.1016/0006-291x(90)90829-c. [DOI] [PubMed] [Google Scholar]
  33. Suter P., Rosenbusch J. P. Asymmetry of binding and physical assignments of CTP and ATP sites in aspartate transcarbamoylase. J Biol Chem. 1977 Nov 25;252(22):8136–8141. [PubMed] [Google Scholar]
  34. Thiry L., Hervé G. The stimulation of Escherichia coli aspartate transcarbamylase activity by adenosine triphosphate. Relation with the other regulatory conformational changes; a model. J Mol Biol. 1978 Nov 15;125(4):515–534. doi: 10.1016/0022-2836(78)90314-5. [DOI] [PubMed] [Google Scholar]
  35. Tondre C., Hammes G. G. Interaction of aspartate transcarbamylase with 5-bromocytidine 5'-tri-, di-, and monophosphates. Biochemistry. 1974 Jul 16;13(15):3131–3136. doi: 10.1021/bi00712a020. [DOI] [PubMed] [Google Scholar]
  36. Wallace A. C., Laskowski R. A., Thornton J. M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 1995 Feb;8(2):127–134. doi: 10.1093/protein/8.2.127. [DOI] [PubMed] [Google Scholar]
  37. Weber H., Khorana H. G. CIV. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast. Chemical synthesis of an icosadeoxynucleotide corresponding to the nucleotide sequence 21 to 40. J Mol Biol. 1972 Dec 28;72(2):219–249. doi: 10.1016/0022-2836(72)90147-7. [DOI] [PubMed] [Google Scholar]
  38. Wente S. R., Schachman H. K. Shared active sites in oligomeric enzymes: model studies with defective mutants of aspartate transcarbamoylase produced by site-directed mutagenesis. Proc Natl Acad Sci U S A. 1987 Jan;84(1):31–35. doi: 10.1073/pnas.84.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wild J. R., Loughrey-Chen S. J., Corder T. S. In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase. Proc Natl Acad Sci U S A. 1989 Jan;86(1):46–50. doi: 10.1073/pnas.86.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Williams M. K., Stec B., Kantrowitz E. R. A single mutation in the regulatory chain of Escherichia coli aspartate transcarbamoylase results in an extreme T-state structure. J Mol Biol. 1998 Aug 7;281(1):121–134. doi: 10.1006/jmbi.1998.1923. [DOI] [PubMed] [Google Scholar]
  41. Winlund C. C., Chamberlin M. J. Binding of cytidine triphosphate to aspartate transcarbamylase. Biochem Biophys Res Commun. 1970 Jul 13;40(1):43–49. doi: 10.1016/0006-291x(70)91043-0. [DOI] [PubMed] [Google Scholar]
  42. Zhang Y., Kantrowitz E. R. Lysine-60 in the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for the discrimination between CTP and ATP. Biochemistry. 1989 Sep 5;28(18):7313–7318. doi: 10.1021/bi00444a025. [DOI] [PubMed] [Google Scholar]
  43. Zhang Y., Kantrowitz E. R. Probing the regulatory site of Escherichia coli aspartate transcarbamoylase by site-specific mutagenesis. Biochemistry. 1992 Jan 28;31(3):792–798. doi: 10.1021/bi00118a022. [DOI] [PubMed] [Google Scholar]
  44. Zhang Y., Kantrowitz E. R. The synergistic inhibition of Escherichia coli aspartate carbamoyltransferase by UTP in the presence of CTP is due to the binding of UTP to the low affinity CTP sites. J Biol Chem. 1991 Nov 25;266(33):22154–22158. [PubMed] [Google Scholar]

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