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
. 1978 Jul;75(7):3138–3142. doi: 10.1073/pnas.75.7.3138

Allosteric regulation of monocyclic interconvertible enzyme cascade systems: Use of Escherichia coli glutamine synthetase as an experimental model

S G Rhee 1, R Park 1,*, P B Chock 1, E R Stadtman 1
PMCID: PMC392729  PMID: 28522

Abstract

The interconversion of Escherichia coli glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] between its adenylylated and unadenylylated forms has been used to verify the prediction derived from a theoretical analysis of the steady-state functions of a model for a monocyclic interconvertible enzyme cascade system [Stadtman, E. R. & Chock, P. B. (1977) Proc. Natl. Acad. Sci. USA 74, 2761-2770]. Because glutamine and α-ketoglutarate are multifunctional effectors and because three active enzyme complexes are involved in both adenylylation and deadenylylation of glutamine synthetase, at least 28 constants are required to describe the glutamine synthetase monocyclic cascade. Of these, 22 constants were determined experimentally and 6 were estimated via computer curve fitting. Despite the complexity, when both adenylylation and deadenylylation reactions are functioning, the number of adenylyl groups bound per mole of enzyme, n̄, assumes a steady-state level as is predicted by the model. This n̄ value is determined by the mole fraction of PIIA—given by ([PIIA]/([PIIA] + [PIID])—and the ratio of glutamine to α-ketoglutarate (PIID and PIID are the unmodified and the uridylylated forms of the PII regulatory protein). In the presence of 0.5 mM glutamine and 2 mM α-ketoglutarate, the value of n̄ increases as a nearly hyperbolic function in response to increasing mole fractions of PIIA. When the constant level of α-ketoglutarate is gradually increased to 40 μM, the hyperbolic function converts slowly to a parabolic function. When the PIIA mole fraction was maintained at 0.6 and α-ketoglutarate levels were varied from 1 mM to 4 μM, an 800-fold increase in signal amplification was observed with respect to glutamine activation. In addition, because glutamine activates the adenylylation and inhibits the deadenylylation reaction, a sensitivity index of 2.1 (corresponding to a Hill number of 1.5) was obtained for the variation of n̄ values in response to increasing glutamine concentration.

Keywords: cyclic cascade, covalent modification, steady state, metabolic regulation

Full text

PDF
3138

Selected References

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

  1. Adler S. P., Purich D., Stadtman E. R. Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme. J Biol Chem. 1975 Aug 25;250(16):6264–6272. [PubMed] [Google Scholar]
  2. Chock P. B., Stadtman E. R. Superiority of interconvertible enzyme cascades in metabolite regulation: analysis of multicyclic systems. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2766–2770. doi: 10.1073/pnas.74.7.2766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Foor F., Janssen K. A., Magasanik B. Regulation of synthesis of glutamine synthetase by adenylylated glutamine synthetase. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4844–4848. doi: 10.1073/pnas.72.12.4844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Greengard P. Phosphorylated proteins as physiological effectors. Science. 1978 Jan 13;199(4325):146–152. doi: 10.1126/science.22932. [DOI] [PubMed] [Google Scholar]
  5. Hennig S. B., Ginsburg A. ATP: glutamine synthetase adenylytransferase from Escherichia coli: purification and properties of a low-molecular weight enzyme form. Arch Biochem Biophys. 1971 Jun;144(2):611–627. doi: 10.1016/0003-9861(71)90368-7. [DOI] [PubMed] [Google Scholar]
  6. Mangum J. H., Magni G., Stadtman E. R. Regulation of glutamine synthetase adenylylation and deadenylylation by the enzymatic uridylylation and deuridylylation of the PII regulatory protein. Arch Biochem Biophys. 1973 Oct;158(2):514–525. doi: 10.1016/0003-9861(73)90543-2. [DOI] [PubMed] [Google Scholar]
  7. Rhee S. G., Chock P. B., Stadtman E. R. Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reaction. Biochimie. 1976;58(1-2):35–49. doi: 10.1016/s0300-9084(76)80354-9. [DOI] [PubMed] [Google Scholar]
  8. Shapiro B. M., Kingdon H. S., Stadtman E. R. Regulation of glutamine synthetase. VII. Adenylyl glutamine synthetase: a new form of the enzyme with altered regulatory and kinetic properties. Proc Natl Acad Sci U S A. 1967 Aug;58(2):642–649. doi: 10.1073/pnas.58.2.642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Stadtman E. R., Chock P. B. Superiority of interconvertible enzyme cascades in metabolic regulation: analysis of monocyclic systems. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2761–2765. doi: 10.1073/pnas.74.7.2761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Villafranca J. J., Balakrishnan M. S., Wedler F. C. Determination of metal-metal distances in E. coli glutamine synthetase by EPR. Biochem Biophys Res Commun. 1977 Mar 21;75(2):464–471. doi: 10.1016/0006-291x(77)91065-8. [DOI] [PubMed] [Google Scholar]
  11. Woolfolk C. A., Shapiro B., Stadtman E. R. Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch Biochem Biophys. 1966 Sep 26;116(1):177–192. doi: 10.1016/0003-9861(66)90026-9. [DOI] [PubMed] [Google Scholar]
  12. Wulff K., Mecke D., Holzer H. Mechanism of the enzymatic inactivation of glutamine synthetase from E. coli. Biochem Biophys Res Commun. 1967 Sep 7;28(5):740–745. doi: 10.1016/0006-291x(67)90378-6. [DOI] [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