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. 1983 Jul 1;213(1):187–191. doi: 10.1042/bj2130187

Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli.

A Lewendon, J R Coggins
PMCID: PMC1152107  PMID: 6351837

Abstract

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

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Selected References

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

  1. Ahmed S. I., Giles N. H. Organization of enzymes in the common aromatic synthetic pathway: evidence for aggregation in fungi. J Bacteriol. 1969 Jul;99(1):231–237. doi: 10.1128/jb.99.1.231-237.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berlyn M. B., Ahmed S. I., Giles N. H. Organization of polyaromatic biosynthetic enzymes in a variety of photosynthetic organisms. J Bacteriol. 1970 Nov;104(2):768–774. doi: 10.1128/jb.104.2.768-774.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berlyn M. B., Giles N. H. Organization of enzymes in the polyaromatic synthetic pathway: separability in bacteria. J Bacteriol. 1969 Jul;99(1):222–230. doi: 10.1128/jb.99.1.222-230.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bode R., Birnbaum D. Aggregation und Trennbarkeit der Enzyme des Shikimat-Pathway bei Hefen. Z Allg Mikrobiol. 1981;21(6):417–422. doi: 10.1002/jobm.3630210602. [DOI] [PubMed] [Google Scholar]
  5. Bondinell W. E., Vnek J., Knowles P. F., Sprecher M., Sprinson D. B. On the mechanism of 5-enolpyruvylshikimate 3-phosphate synthetase. J Biol Chem. 1971 Oct 25;246(20):6191–6196. [PubMed] [Google Scholar]
  6. Boocock M. R., Coggins J. R. Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate. FEBS Lett. 1983 Apr 5;154(1):127–133. doi: 10.1016/0014-5793(83)80888-6. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Cassidy P. J., Kahan F. M. A stable enzyme-phosphoenolpyruvate intermediate in the synthesis of uridine-5'-diphospho-N-acetyl-2-amino-2-deoxyglucose 3-O-enolpyruvyl ether. Biochemistry. 1973 Mar 27;12(7):1364–1374. doi: 10.1021/bi00731a017. [DOI] [PubMed] [Google Scholar]
  9. Castellino F. J., Barker R. Examination of the dissociation of multichain proteins in guanidine hydrochloride by membrane osmometry. Biochemistry. 1968 Jun;7(6):2207–2217. doi: 10.1021/bi00846a025. [DOI] [PubMed] [Google Scholar]
  10. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  11. ENGSTROM L. Studies on calf-intestinal alkaline phosphatase. I. Chromatographic purification, microheterogeneity and some other properties of the purified enzyme. Biochim Biophys Acta. 1961 Sep 2;52:36–48. doi: 10.1016/0006-3002(61)90901-5. [DOI] [PubMed] [Google Scholar]
  12. Gaertner F. H., Cole K. W. A cluster-gene: evidence for one gene, one polypeptide, five enzymes. Biochem Biophys Res Commun. 1977 Mar 21;75(2):259–264. doi: 10.1016/0006-291x(77)91037-3. [DOI] [PubMed] [Google Scholar]
  13. Grimshaw C. E., Sogo S. G., Knowles J. R. The fate of the hydrogens of phosphoenolpyruvate in the reaction catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase. Isotope effects and isotope exchange. J Biol Chem. 1982 Jan 25;257(2):596–598. [PubMed] [Google Scholar]
  14. Gunetileke K. G., Anwar R. A. Biosynthesis of uridine diphospho-N-acetylmuramic acid. II. Purification and properties of pyruvate-uridine diphospho-N-acetylglucosamine transferase and characterization of uridine diphospho-N-acetylenopyruvylglucosamine. J Biol Chem. 1968 Nov 10;243(21):5770–5778. [PubMed] [Google Scholar]
  15. Ife R. J., Ball L. F., Lowe P., Haslam E. The shikimate pathway. Part V. Chorismic acid and chorismate mutase. J Chem Soc Perkin 1. 1976;(16):1776–1783. doi: 10.1039/p19760001776. [DOI] [PubMed] [Google Scholar]
  16. Kahan F. M., Kahan J. S., Cassidy P. J., Kropp H. The mechanism of action of fosfomycin (phosphonomycin). Ann N Y Acad Sci. 1974 May 10;235(0):364–386. doi: 10.1111/j.1749-6632.1974.tb43277.x. [DOI] [PubMed] [Google Scholar]
  17. Krietsch W. K., Bücher T. 3-phosphoglycerate kinase from rabbit sceletal muscle and yeast. Eur J Biochem. 1970 Dec;17(3):568–580. doi: 10.1111/j.1432-1033.1970.tb01202.x. [DOI] [PubMed] [Google Scholar]
  18. LEVIN J. G., SPRINSON D. B. THE ENZYMATIC FORMATION AND ISOLATION OF 3-ENOLPYRUVYLSHIKIMATE 5-PHOSPHATE. J Biol Chem. 1964 Apr;239:1142–1150. [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lumsden J., Coggins J. R. The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain. Biochem J. 1977 Mar 1;161(3):599–607. doi: 10.1042/bj1610599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. MARGOLIASH E., SMITH E. L., KREIL G., TUPPY H. Amino-acid sequence of horse heart cytochrome c. Nature. 1961 Dec 23;192:1125–1127. doi: 10.1038/1921125a0. [DOI] [PubMed] [Google Scholar]
  22. Nimmo H. G., Nimmo G. A. A general method for the localization of enzymes that produce phosphate, pyrophosphate, or CO2 after polyacrylamide gel electrophoresis. Anal Biochem. 1982 Mar 15;121(1):17–22. doi: 10.1016/0003-2697(82)90551-6. [DOI] [PubMed] [Google Scholar]
  23. Patel V. B., Giles N. H. Purification of the arom multienzyme aggregate from Euglena gracilis. Biochim Biophys Acta. 1979 Mar 16;567(1):24–34. doi: 10.1016/0005-2744(79)90168-2. [DOI] [PubMed] [Google Scholar]
  24. Shaltiel S. Hydrophobic chromatography. Methods Enzymol. 1974;34:126–140. doi: 10.1016/s0076-6879(74)34012-8. [DOI] [PubMed] [Google Scholar]
  25. Steinmetz M. A., Deal W. C., Jr Metabolic control and structure of glycolytic enzymes. 3. Dissociation and subunit structure of rabbit muscle pyruvate kinase. Biochemistry. 1966 Apr;5(4):1399–1405. doi: 10.1021/bi00868a038. [DOI] [PubMed] [Google Scholar]
  26. Steinrücken H. C., Amrhein N. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase. Biochem Biophys Res Commun. 1980 Jun 30;94(4):1207–1212. doi: 10.1016/0006-291x(80)90547-1. [DOI] [PubMed] [Google Scholar]
  27. THORNE C. J., KAPLAN N. O. Physicochemical properties of pig and horse heart mitochondrial malate dehydrogenase. J Biol Chem. 1963 May;238:1861–1868. [PubMed] [Google Scholar]
  28. Zemell R. I., Anwar R. A. Mechanism of pyruvate-uridine diphospho-N-acetylglucosamine transferase. Evidence for an enzyme-enolpyruvate intermediate. J Biol Chem. 1975 Jul 10;250(13):4959–4964. [PubMed] [Google Scholar]

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