Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1979 Aug;76(8):3732–3736. doi: 10.1073/pnas.76.8.3732

Communication between dissimilar subunits in aspartate transcarbamoylase: Effect of inhibitor and activator on the conformation of the catalytic polypeptide chains

Preston Hensley 1, H K Schachman 1,*
PMCID: PMC383907  PMID: 386346

Abstract

Although local, direct effects of ligand binding to proteins are readily differentiated conceptually from gross, indirect conformational changes in regions of the protein remote from the site of binding, it has been difficult experimentally to distinguish between them. In oligomeric proteins, for example, the binding of ligands to one chain may cause a conformational change in the unliganded chains, but many physical chemical probes are not sufficiently discriminating to demonstrate where the change occurred. Evidence has been lacking as to whether the inhibitor, CTP, or the activator, ATP, in binding to the regulatory chains of the allosteric enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) causes a conformational change that is propagated throughout the enzyme to the catalytic chains. To demonstrate this “communication” of effects of binding at one site on the conformation of other polypeptide chains, we constructed molecules containing native regulatory subunits and enzymically active nitrated catalytic subunits having one sensitive nitrotyrosyl chromophore per polypeptide chain. These hybrid molecules exhibited the characteristic regulatory properties of the native enzyme. Upon the addition of CTP the population of molecules was shifted toward the constrained or T state, as shown by the change in the sedimentation coefficient and the altered enzyme kinetics. Moreover, there was a decrease in absorbance at 430 nm due to the altered environment of the nitrated catalytic polypeptide chains. In contrast, ATP caused a shift toward the relaxed or R conformation, and the absorbance due to the nitrotyrosyl residues was increased. Different types of experiments indicated that the modified enzyme molecules are in a preexisting equilibrium that is perturbed by CTP or ATP; the resulting conformational changes in the nitrated catalytic subunits are detected by opposite alterations in their absorbance spectrum.

Keywords: allosteric enzymes, subunit interactions, cooperativity, intersubunit hybrids, aspartate carbamoyltransferase

Full text

PDF
3732

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blackburn M. N., Schachman H. K. Allosteric regulation of aspartate transcarbamoylase. Effect of active site ligands on the reactivity of sulfhydryl groups of the regulatory subunits. Biochemistry. 1977 Nov 15;16(23):5084–5091. doi: 10.1021/bi00642a022. [DOI] [PubMed] [Google Scholar]
  2. Buckman T. Spin-labeling studies of aspartate transcarbamylase. I. Effects of nucleotide binding and subunit separation. Biochemistry. 1970 Aug 4;9(16):3255–3265. doi: 10.1021/bi00818a020. [DOI] [PubMed] [Google Scholar]
  3. Cohlberg J. A., Pigiet V. P., Jr, Schachman H. K. Structure and arrangement of the regulatory subunits in aspartate transcarbamylase. Biochemistry. 1972 Aug 29;11(18):3396–3411. doi: 10.1021/bi00768a013. [DOI] [PubMed] [Google Scholar]
  4. Collins K. D., Stark G. R. Aspartate transcarbamylase. Interaction with the transition state analogue N-(phosphonacetyl)-L-aspartate. J Biol Chem. 1971 Nov;246(21):6599–6605. [PubMed] [Google Scholar]
  5. Davies G. E., Vanaman T. C., Stark G. R. Aspartate transcarbamylase. Stereospecific restrictions on the binding site for L-aspartate. J Biol Chem. 1970 Mar 10;245(5):1175–1179. [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., Schachman H. K. Allosteric interactions in aspartate transcarbamylase. II. Evidence for different conformational states of the protein in the presence and absence of specific ligands. Biochemistry. 1968 Feb;7(2):538–552. doi: 10.1021/bi00842a600. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Griffin J. H., Rosenbusch J. P., Blout E. R., Weber K. K. Conformational changes in aspartate transcarbamylase. II. Circular dichroism evidence for the involvement of metal ions in allosteric interactions. J Biol Chem. 1973 Jul 25;248(14):5057–5062. [PubMed] [Google Scholar]
  10. Hahn L. H., Hammes G. G. Structural mapping of aspartate transcarbamoylase by fluorescence energy-transfer measurements: determination of the distance between catalytic sites of different subunits. Biochemistry. 1978 Jun 13;17(12):2423–2429. doi: 10.1021/bi00605a027. [DOI] [PubMed] [Google Scholar]
  11. Harrison L. W., Hammes G. G. Relaxation spectra of aspartate transcarbamylase. Interaction of the native enzyme with cytidine 5'-triphosphate. Biochemistry. 1973 Mar 27;12(7):1395–1400. doi: 10.1021/bi00731a020. [DOI] [PubMed] [Google Scholar]
  12. Howlett G. J., Blackburn M. N., Compton J. G., Schachman H. K. Allosteric regulation of aspartate transcarbamoylase. Analysis of the structural and functional behavior in terms of a two-state model. Biochemistry. 1977 Nov 15;16(23):5091–5100. doi: 10.1021/bi00642a023. [DOI] [PubMed] [Google Scholar]
  13. Howlett G. J., Schachman H. K. Allosteric regulation of aspartate transcarbamoylase. Changes in the sedimentation coefficient promoted by the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Biochemistry. 1977 Nov 15;16(23):5077–5083. doi: 10.1021/bi00642a021. [DOI] [PubMed] [Google Scholar]
  14. Kempe T. D., Stark G. R. Pyridoxal 5'-phosphate, a fluorescent probe in the active site of aspartate transcarbamylase. J Biol Chem. 1975 Sep 10;250(17):6861–6869. [PubMed] [Google Scholar]
  15. Kirschner M. W., Schachman H. K. Conformational studies on the nitrated catalytic subunit of aspartate transcarbamylase. Biochemistry. 1973 Jul 31;12(16):2987–2997. doi: 10.1021/bi00740a007. [DOI] [PubMed] [Google Scholar]
  16. Kirschner M. W., Schachman H. K. Local and gross conformational changes in aspartate transcarbamylase. Biochemistry. 1973 Jul 31;12(16):2997–3004. doi: 10.1021/bi00740a008. [DOI] [PubMed] [Google Scholar]
  17. Koshland D. E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966 Jan;5(1):365–385. doi: 10.1021/bi00865a047. [DOI] [PubMed] [Google Scholar]
  18. Landfear S. M., Evans D. R., Lipscomb W. N. Elimination of cooperativity in aspartate transcarbamylase by nitration of a single tyrosine residue. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2654–2658. doi: 10.1073/pnas.75.6.2654. [DOI] [PMC free article] [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. Meighen E. A., Pigiet V., Schachman H. K. Hybridization of native and chemically modified enzymes. 3. The catalytic subunits of aspartate transcarbamylase. Proc Natl Acad Sci U S A. 1970 Jan;65(1):234–241. doi: 10.1073/pnas.65.1.234. [DOI] [PMC free article] [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. Ogawa S., Shulman R. G. High resolution nuclear magnetic resonance spectra of hemoglobin. 3. The half-ligated state and allosteric interactions. J Mol Biol. 1972 Sep 28;70(2):315–336. doi: 10.1016/0022-2836(72)90542-6. [DOI] [PubMed] [Google Scholar]
  23. Perutz M. F. Nature of haem-haem interaction. Nature. 1972 Jun 30;237(5357):495–499. doi: 10.1038/237495a0. [DOI] [PubMed] [Google Scholar]
  24. Perutz M. F., Pulsinelli P. D., Ranney H. M. Structure and subunit interaction of haemoglobin M Milwaukee. Nat New Biol. 1972 Jun 28;237(78):259–263. doi: 10.1038/newbio237259a0. [DOI] [PubMed] [Google Scholar]
  25. Porter R. W., Modebe M. O., Stark G. R. Aspartate transcarbamylase. Kinetic studies of the catalytic subunit. J Biol Chem. 1969 Apr 10;244(7):1846–1859. [PubMed] [Google Scholar]
  26. Riordan J. F., Sokolovsky M., Vallee B. L. Environmentally sensitive tyrosyl residues. Nitration with tetranitromethane. Biochemistry. 1967 Jan;6(1):358–361. doi: 10.1021/bi00853a053. [DOI] [PubMed] [Google Scholar]
  27. Rosenbusch J. P., Weber K. Subunit structure of aspartate transcarbamylase from Escherichia coli. J Biol Chem. 1971 Mar 25;246(6):1644–1657. [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Weber K. New structural model of E. coli aspartate transcarbamylase and the amino-acid sequence of the regulatory polypeptide chain. Nature. 1968 Jun 22;218(5147):1116–1119. doi: 10.1038/2181116a0. [DOI] [PubMed] [Google Scholar]
  31. Wiley D. C., Lipscomb W. N. Crystallographic determination of symmetry of aspartate transcarbamylase. Nature. 1968 Jun 22;218(5147):1119–1121. doi: 10.1038/2181119a0. [DOI] [PubMed] [Google Scholar]
  32. Yang Y. R., Kirschner M. W., Schachman H. K. Aspartate transcarbamoylase (Escherichia coli): preparation of subunits. Methods Enzymol. 1978;51:35–41. doi: 10.1016/s0076-6879(78)51007-0. [DOI] [PubMed] [Google Scholar]
  33. Yang Y. R., Syvanen J. M., Nagel G. M., Schachman H. K. Aspartate transcarbamoylase molecules lacking one regulatory subunit. Proc Natl Acad Sci U S A. 1974 Mar;71(3):918–922. doi: 10.1073/pnas.71.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES