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. 1998 Jan 15;329(Pt 2):243–247. doi: 10.1042/bj3290243

Aspartate-90 and arginine-269 of hamster aspartate transcarbamylase affect the oligomeric state of a chimaeric protein with an Escherichia coli maltose-binding domain.

Y Qiu 1, J N Davidson 1
PMCID: PMC1219037  PMID: 9425105

Abstract

Residues Asp-90 and Arg-269 of Escherichia coli aspartate transcarbamylase seem to interact at the interface of adjacent catalytic subunits. Alanine substitutions at the analogous positions in the hamster aspartate transcarbamylase of a chimaeric protein carrying an E. coli maltose-binding domain lead to changes in both the kinetics of the enzyme and the quaternary structure of the protein. The Vmax for the Asp-90-->Ala and Arg-269-->Ala substitutions is decreased to 1/21 and 1/50 respectively, the [S]0.5 for aspartate is increased 540-fold and 826-fold respectively, and the [S]0.5 for carbamoyl phosphate is increased 60-fold for both. These substitutions decrease the oligomeric size of the protein. Whereas the native chimaeric protein behaves as a pentamer, the Asp-90 variant is a trimer and the Arg-269 variant is a dimer. The altered enzymes also exhibit marked decreases in thermal stability and are inactivated at much lower concentrations of urea than is the unaltered enzyme. Taken together, these results are consistent with the hypothesis that both Asp-90 and Arg-269 have a role in the enzymic function and structural integrity of hamster aspartate transcarbamylase.

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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. Beck D., Kedzie K. M., Wild J. R. Comparison of the aspartate transcarbamoylases from Serratia marcescens and Escherichia coli. J Biol Chem. 1989 Oct 5;264(28):16629–16637. [PubMed] [Google Scholar]
  3. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Coleman P. F., Suttle D. P., Stark G. R. Purification from hamster cells of the multifunctional protein that initiates de novo synthesis of pyrimidine nucleotides. J Biol Chem. 1977 Sep 25;252(18):6379–6385. [PubMed] [Google Scholar]
  5. Davidson D. M., Smith R. M., Qaqundah P. Y. Cholesterol screening in children during office visits. J Pediatr Health Care. 1990 Jan-Feb;4(1):11–17. doi: 10.1016/0891-5245(90)90034-4. [DOI] [PubMed] [Google Scholar]
  6. Davidson J. N., Rao G. N., Niswander L., Andreano C., Tamer C., Chen K. C. Organization and nucleotide sequence of the 3' end of the human CAD gene. DNA Cell Biol. 1990 Nov;9(9):667–676. doi: 10.1089/dna.1990.9.667. [DOI] [PubMed] [Google Scholar]
  7. Davidson J. N., Rumsby P. C., Tamaren J. Organization of a multifunctional protein in pyrimidine biosynthesis. Analyses of active, tryptic fragments. J Biol Chem. 1981 May 25;256(10):5220–5225. [PubMed] [Google Scholar]
  8. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  9. Faure M., Camonis J. H., Jacquet M. Molecular characterization of a Dictyostelium discoideum gene encoding a multifunctional enzyme of the pyrimidine pathway. Eur J Biochem. 1989 Feb 1;179(2):345–358. doi: 10.1111/j.1432-1033.1989.tb14560.x. [DOI] [PubMed] [Google Scholar]
  10. Freund J. N., Jarry B. P. The rudimentary gene of Drosophila melanogaster encodes four enzymic functions. J Mol Biol. 1987 Jan 5;193(1):1–13. doi: 10.1016/0022-2836(87)90621-8. [DOI] [PubMed] [Google Scholar]
  11. Guy H. I., Evans D. R. Cloning and expression of the mammalian multifunctional protein CAD in Escherichia coli. Characterization of the recombinant protein and a deletion mutant lacking the major interdomain linker. J Biol Chem. 1994 Sep 23;269(38):23808–23816. [PubMed] [Google Scholar]
  12. Hemmens B., Carrey E. A. Mammalian dihydroorotase; secondary structure, and interactions with other proteolytic fragments from the multienzyme polypeptide CAD. Eur J Biochem. 1995 Jul 1;231(1):220–225. [PubMed] [Google Scholar]
  13. Hoover T. A., Roof W. D., Foltermann K. F., O'Donovan G. A., Bencini D. A., Wild J. R. Nucleotide sequence of the structural gene (pyrB) that encodes the catalytic polypeptide of aspartate transcarbamoylase of Escherichia coli. Proc Natl Acad Sci U S A. 1983 May;80(9):2462–2466. doi: 10.1073/pnas.80.9.2462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim H., Kelly R. E., Evans D. R. The structural organization of the hamster multifunctional protein CAD. Controlled proteolysis, domains, and linkers. J Biol Chem. 1992 Apr 5;267(10):7177–7184. [PubMed] [Google Scholar]
  15. Lerner C. G., Switzer R. L. Cloning and structure of the Bacillus subtilis aspartate transcarbamylase gene (pyrB). J Biol Chem. 1986 Aug 25;261(24):11156–11165. [PubMed] [Google Scholar]
  16. 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]
  17. Major J. G., Jr, Wales M. E., Houghton J. E., Maley J. A., Davidson J. N., Wild J. R. Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase. J Mol Evol. 1989 May;28(5):442–450. doi: 10.1007/BF02603079. [DOI] [PubMed] [Google Scholar]
  18. Maley J. A., Davidson J. N. The aspartate transcarbamylase domain of a mammalian multifunctional protein expressed as an independent enzyme in Escherichia coli. Mol Gen Genet. 1988 Aug;213(2-3):278–284. doi: 10.1007/BF00339592. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Patterson D., Carnright D. V. Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: I. Isolation of a mutant defective in the early steps of de novo pyrimidine synthesis. Somatic Cell Genet. 1977 Sep;3(5):483–495. doi: 10.1007/BF01539120. [DOI] [PubMed] [Google Scholar]
  21. Prescott L. M., Jones M. E. Modified methods for the determination of carbamyl aspartate. Anal Biochem. 1969 Dec;32(3):408–419. doi: 10.1016/s0003-2697(69)80008-4. [DOI] [PubMed] [Google Scholar]
  22. Richarme G. Associative properties of the Escherichia coli galactose binding protein and maltose binding protein. Biochem Biophys Res Commun. 1982 Mar 30;105(2):476–481. doi: 10.1016/0006-291x(82)91459-0. [DOI] [PubMed] [Google Scholar]
  23. Shanley M. S., Foltermann K. F., O'Donovan G. A., Wild J. R. Properties of hybrid aspartate transcarbamoylase formed with native subunits from divergent bacteria. J Biol Chem. 1984 Oct 25;259(20):12672–12677. [PubMed] [Google Scholar]
  24. Shigesada K., Stark G. R., Maley J. A., Niswander L. A., Davidson J. N. Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase. Mol Cell Biol. 1985 Jul;5(7):1735–1742. doi: 10.1128/mcb.5.7.1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Souciet J. L., Nagy M., Le Gouar M., Lacroute F., Potier S. Organization of the yeast URA2 gene: identification of a defective dihydroorotase-like domain in the multifunctional carbamoylphosphate synthetase-aspartate transcarbamylase complex. Gene. 1989 Jun 30;79(1):59–70. doi: 10.1016/0378-1119(89)90092-9. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Williamson C. L., Slocum R. D. Molecular cloning and characterization of the pyrB1 and pyrB2 genes encoding aspartate transcarbamoylase in pea (Pisum sativum L.). Plant Physiol. 1994 May;105(1):377–384. doi: 10.1104/pp.105.1.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yang Y. R., Schachman H. K. A bifunctional fusion protein containing the maltose-binding polypeptide and the catalytic chain of aspartate transcarbamoylase: assembly, oligomers, and domains. Biophys Chem. 1996 Apr 16;59(3):289–297. doi: 10.1016/0301-4622(96)00018-x. [DOI] [PubMed] [Google Scholar]
  30. di Guan C., Li P., Riggs P. D., Inouye H. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene. 1988 Jul 15;67(1):21–30. doi: 10.1016/0378-1119(88)90004-2. [DOI] [PubMed] [Google Scholar]

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