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. 1993 Jan;2(1):103–112. doi: 10.1002/pro.5560020111

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

B B Zhou 1, H K Schachman 1
PMCID: PMC2142301  PMID: 8443583

Abstract

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.

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Selected References

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

  1. Bethell M. R., Smith K. E., White J. S., Jones M. E. Carbamyl phosphate: an allosteric substrate for aspartate transcarbamylase of Escherichia coli. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1442–1449. doi: 10.1073/pnas.60.4.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Eisenstein E., Markby D. W., Schachman H. K. Changes in stability and allosteric properties of aspartate transcarbamoylase resulting from amino acid substitutions in the zinc-binding domain of the regulatory chains. Proc Natl Acad Sci U S A. 1989 May;86(9):3094–3098. doi: 10.1073/pnas.86.9.3094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eisenstein E., Markby D. W., Schachman H. K. Heterotropic effectors promote a global conformational change in aspartate transcarbamoylase. Biochemistry. 1990 Apr 17;29(15):3724–3731. doi: 10.1021/bi00467a019. [DOI] [PubMed] [Google Scholar]
  7. GERHART J. C., PARDEE A. B. The enzymology of control by feedback inhibition. J Biol Chem. 1962 Mar;237:891–896. [PubMed] [Google Scholar]
  8. 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]
  9. Gerhart J. C., Schachman H. K. Distinct subunits for the regulation and catalytic activity of aspartate transcarbamylase. Biochemistry. 1965 Jun;4(6):1054–1062. doi: 10.1021/bi00882a012. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Kantrowitz E. R., Lipscomb W. N. Escherichia coli aspartate transcarbamylase: the relation between structure and function. Science. 1988 Aug 5;241(4866):669–674. doi: 10.1126/science.3041592. [DOI] [PubMed] [Google Scholar]
  12. Ke H. M., Lipscomb W. N., Cho Y. J., Honzatko R. B. Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase. X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms. J Mol Biol. 1988 Dec 5;204(3):725–747. doi: 10.1016/0022-2836(88)90365-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Markby D. W., Zhou B. B., Schachman H. K. A 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase forms a stable complex with the catalytic subunit leading to markedly altered enzyme activity. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10568–10572. doi: 10.1073/pnas.88.23.10568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  17. Newell J. O., Markby D. W., Schachman H. K. Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP. J Biol Chem. 1989 Feb 15;264(5):2476–2481. [PubMed] [Google Scholar]
  18. Newell J. O., Schachman H. K. Amino acid substitutions which stabilize aspartate transcarbamoylase in the R state disrupt both homotropic and heterotropic effects. Biophys Chem. 1990 Aug 31;37(1-3):183–196. doi: 10.1016/0301-4622(90)88018-n. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Peterson C. B., Burman D. L., Schachman H. K. Effects of replacement of active site residue glutamine 231 on activity and allosteric properties of aspartate transcarbamoylase. Biochemistry. 1992 Sep 15;31(36):8508–8515. doi: 10.1021/bi00151a018. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Subramani S., Bothwell M. A., Gibbons I., Yang Y. R., Schachman H. K. Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamolylase. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3777–3781. doi: 10.1073/pnas.74.9.3777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wall K. A., Flatgaard J. E., Schachman H. K., Gibbons I. Purification and characterization of a mutant aspartate transcarbamoylase lacking enzyme activity. J Biol Chem. 1979 Dec 10;254(23):11910–11916. [PubMed] [Google Scholar]
  24. 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]

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