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. 1999 Jun;8(6):1305–1313. doi: 10.1110/ps.8.6.1305

The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity.

C Macol 1, M Dutta 1, B Stec 1, H Tsuruta 1, E R Kantrowitz 1
PMCID: PMC2144362  PMID: 10386880

Abstract

The X-ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl-L-aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site-specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild-type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild-type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5- to 10-fold, and the affinity for carbamoyl phosphate was reduced 10- to 30-fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild-type activity, while the K83A enzyme exhibited 61% of wild-type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high-activity, high-affinity active site. Since low-angle X-ray scattering demonstrated that the mutant enzymes can be converted to the R-structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high-activity, high-affinity active site characteristic of the R-functional state of the enzyme.

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Selected References

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  1. Baker D. P., Kantrowitz E. R. The conserved residues glutamate-37, aspartate-100, and arginine-269 are important for the structural stabilization of Escherichia coli aspartate transcarbamoylase. Biochemistry. 1993 Sep 28;32(38):10150–10158. doi: 10.1021/bi00089a034. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [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. Collins K. D., Stark G. R. Aspartate transcarbamylase. Studies of the catalytic subunit by ultraviolet difference spectroscopy. J Biol Chem. 1969 Apr 10;244(7):1869–1877. [PubMed] [Google Scholar]
  5. Fetler L., Tauc P., Hervé G., Moody M. F., Vachette P. X-ray scattering titration of the quaternary structure transition of aspartate transcarbamylase with a bisubstrate analogue: influence of nucleotide effectors. J Mol Biol. 1995 Aug 11;251(2):243–255. doi: 10.1006/jmbi.1995.0432. [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. Gouaux J. E., Krause K. L., Lipscomb W. N. The catalytic mechanism of Escherichia coli aspartate carbamoyltransferase: a molecular modelling study. Biochem Biophys Res Commun. 1987 Feb 13;142(3):893–897. doi: 10.1016/0006-291x(87)91497-5. [DOI] [PubMed] [Google Scholar]
  8. Gouaux J. E., Lipscomb W. N. Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-A resolution and neutral pH. Biochemistry. 1990 Jan 16;29(2):389–402. doi: 10.1021/bi00454a013. [DOI] [PubMed] [Google Scholar]
  9. Gouaux J. E., Lipscomb W. N. Three-dimensional structure of carbamoyl phosphate and succinate bound to aspartate carbamoyltransferase. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4205–4208. doi: 10.1073/pnas.85.12.4205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Greenwell P., Jewett S. L., Stark G. R. Aspartate transcarbamylase from Escherichia coli. The use of pyridoxal 5'-phosphate as a probe in the active site. J Biol Chem. 1973 Sep 10;248(17):5994–6001. [PubMed] [Google Scholar]
  11. Griffin J. H., Rosenbusch J. P., Weber K. K., Blout E. R. Conformational changes in aspartate trancarbamylase. I. Studies of ligand binding and of subunit interactions by circular dichroism spectroscopy. J Biol Chem. 1972 Oct 25;247(20):6482–6490. [PubMed] [Google Scholar]
  12. Hervé G., Moody M. F., Tauc P., Vachette P., Jones P. T. Quaternary structure changes in aspartate transcarbamylase studied by X-ray solution scattering. Signal transmission following effector binding. J Mol Biol. 1985 Sep 5;185(1):189–199. doi: 10.1016/0022-2836(85)90190-1. [DOI] [PubMed] [Google Scholar]
  13. Hsuanyu Y., Wedler F. C. Kinetic mechanism of native Escherichia coli aspartate transcarbamylase. Arch Biochem Biophys. 1987 Dec;259(2):316–330. doi: 10.1016/0003-9861(87)90498-x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kantrowitz E. R., Lipscomb W. N. Functionally important arginine residues of aspartate transcarbamylase. J Biol Chem. 1977 May 10;252(9):2873–2880. [PubMed] [Google Scholar]
  16. 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]
  17. Kerbiriou D., Hervé G. Biosynthesis of an aspartate transcarbamylase lacking co-operative interactions. I. Disconnection of homotropic and heterotropic interactions under the influence of 2-thiouracil. J Mol Biol. 1972 Mar 14;64(2):379–392. doi: 10.1016/0022-2836(72)90505-0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Krause K. L., Volz K. W., Lipscomb W. N. Structure at 2.9-A resolution of aspartate carbamoyltransferase complexed with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1643–1647. doi: 10.1073/pnas.82.6.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  22. Ladjimi M. M., Middleton S. A., Kelleher K. S., Kantrowitz E. R. Relationship between domain closure and binding, catalysis, and regulation in Escherichia coli aspartate transcarbamylase. Biochemistry. 1988 Jan 12;27(1):268–276. doi: 10.1021/bi00401a041. [DOI] [PubMed] [Google Scholar]
  23. Lahue R. S., Schachman H. K. The influence of quaternary structure on the active site of an oligomeric enzyme. Catalytic subunit of aspartate transcarbamoylase. J Biol Chem. 1984 Nov 25;259(22):13906–13913. [PubMed] [Google Scholar]
  24. 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]
  25. Landfear S. M., Lipscomb W. N., Evans D. R. Functional modifications of aspartate transcarbamylase induced by nitration with tetranitromethane. J Biol Chem. 1978 Jun 10;253(11):3988–3996. [PubMed] [Google Scholar]
  26. Lauritzen A. M., Lipscomb W. N. Modification of three active site lysine residues in the catalytic subunit of aspartate transcarbamylase by D- and L-bromosuccinate. J Biol Chem. 1982 Feb 10;257(3):1312–1319. [PubMed] [Google Scholar]
  27. 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]
  28. Middleton S. A., Kantrowitz E. R. Function of arginine-234 and aspartic acid-271 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamylase. Biochemistry. 1988 Nov 15;27(23):8653–8660. doi: 10.1021/bi00423a022. [DOI] [PubMed] [Google Scholar]
  29. Middleton S. A., Stebbins J. W., Kantrowitz E. R. A loop involving catalytic chain residues 230-245 is essential for the stabilization of both allosteric forms of Escherichia coli aspartate transcarbamylase. Biochemistry. 1989 Feb 21;28(4):1617–1626. doi: 10.1021/bi00430a029. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Newton C. J., Kantrowitz E. R. Importance of domain closure for homotropic cooperativity in Escherichia coli aspartate transcarbamylase. Biochemistry. 1990 Feb 13;29(6):1444–1451. doi: 10.1021/bi00458a015. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. Robey E. A., Wente S. R., Markby D. W., Flint A., Yang Y. R., Schachman H. K. Effect of amino acid substitutions on the catalytic and regulatory properties of aspartate transcarbamoylase. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5934–5938. doi: 10.1073/pnas.83.16.5934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schachman H. K. Can a simple model account for the allosteric transition of aspartate transcarbamoylase? J Biol Chem. 1988 Dec 15;263(35):18583–18586. [PubMed] [Google Scholar]
  39. 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]
  40. Stebbins J. W., Robertson D. E., Roberts M. F., Stevens R. C., Lipscomb W. N., Kantrowitz E. R. Arginine 54 in the active site of Escherichia coli aspartate transcarbamoylase is critical for catalysis: a site-specific mutagenesis, NMR, and X-ray crystallographic study. Protein Sci. 1992 Nov;1(11):1435–1446. doi: 10.1002/pro.5560011105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Stebbins J. W., Zhang Y., Kantrowitz E. R. Importance of residues Arg-167 and Gln-231 in both the allosteric and catalytic mechanisms of Escherichia coli aspartate transcarbamoylase. Biochemistry. 1990 Apr 24;29(16):3821–3827. doi: 10.1021/bi00468a003. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Wu C. W., Hammes G. G. Relaxation spectra of aspartate transcarbamylase. Interaction of the native enzyme with an adenosine 5'-triphosphate analog. Biochemistry. 1973 Mar 27;12(7):1400–1408. doi: 10.1021/bi00731a021. [DOI] [PubMed] [Google Scholar]
  47. Xu W., Kantrowitz E. R. Function of serine-52 and serine-80 in the catalytic mechanism of Escherichia coli aspartate transcarbamoylase. Biochemistry. 1991 Mar 5;30(9):2535–2542. doi: 10.1021/bi00223a034. [DOI] [PubMed] [Google Scholar]
  48. Xu W., Kantrowitz E. R. Function of threonine-55 in the carbamoyl phosphate binding site of Escherichia coli aspartate transcarbamoylase. Biochemistry. 1989 Dec 26;28(26):9937–9943. doi: 10.1021/bi00452a010. [DOI] [PubMed] [Google Scholar]
  49. Xu W., Pitts M. A., Middleton S. A., Kelleher K. S., Kantrowitz E. R. Propagation of allosteric changes through the catalytic-regulatory interface of Escherichia coli aspartate transcarbamylase. Biochemistry. 1988 Jul 26;27(15):5507–5515. doi: 10.1021/bi00415a018. [DOI] [PubMed] [Google Scholar]

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