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. 1984 Feb;3(2):279–287. doi: 10.1002/j.1460-2075.1984.tb01797.x

The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

A N Lane, C H Paul, K Kirschner
PMCID: PMC557335  PMID: 6425053

Abstract

The alpha subunit is bound with negative cooperativity to the holo beta 2 subunit of tryptophan synthase in phosphate buffer. Thus it is feasible to measure separately the rates of formation both of the stable alpha beta 2 subcomplex from beta 2, and of the mature alpha 2 beta 2 complex from alpha beta 2, using stopped-flow techniques. Addition of each alpha subunit proceeds in two steps; an initial alpha beta protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the alpha beta 2 and alpha 2 beta 2 complexes were measured by trapping released alpha subunit with enzymically inactive reduced beta 2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the beta protomer for the alpha subunit. The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants. Negative cooperativity arises from a weaker initial binding of the second alpha subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-serine and indolepropanol phosphate during the assembly process shows that the beta protomer is already partially activated in the initial alpha beta complex. Full activation is achieved in the slow isomerization reaction. In contrast, the alpha subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction. These observations indicate that the isomerization involves synchronous conformation changes of both alpha and beta protomers.

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Selected References

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  1. Adachi O., Kohn L. D., Miles E. W. Crystalline alpha2 beta2 complexes of tryptophan synthetase of Escherichia coli. A comparison between the native complex and the reconstituted complex. J Biol Chem. 1974 Dec 25;249(24):7756–7763. [PubMed] [Google Scholar]
  2. Bartholmes P., Kirschner K., Gschwind H. P. Cooperative and noncooperative binding of pyridoxal 5'-phosphate to tryptophan synthase from Escherichia coli. Biochemistry. 1976 Oct 19;15(21):4712–4717. doi: 10.1021/bi00666a027. [DOI] [PubMed] [Google Scholar]
  3. Bothwell M. A., Schachman H. K. A model for the assembly of aspartate transcarbamoylase from catalytic and regulatory subunits. J Biol Chem. 1980 Mar 10;255(5):1971–1977. [PubMed] [Google Scholar]
  4. Creighton T. E., Yanofsky C. Association of the alpha and beta-2 subunits of the tryptophan synthetase of Escherichia coli. J Biol Chem. 1966 Feb 25;241(4):980–990. [PubMed] [Google Scholar]
  5. Faeder E. J., Hammes G. G. Kinetic studies of tryptophan synthetase. Interaction of L-serine, indole, and tryptophan with the native enzyme. Biochemistry. 1971 Mar 16;10(6):1041–1045. doi: 10.1021/bi00782a016. [DOI] [PubMed] [Google Scholar]
  6. Faeder E. J., Hammes G. G. Kinetic studies of tryptophan synthetase. Interaction of substrates with the B subunit. Biochemistry. 1970 Oct 13;9(21):4043–4049. doi: 10.1021/bi00823a003. [DOI] [PubMed] [Google Scholar]
  7. Friedman F. K., Beychok S. Probes of subunit assembly and reconstitution pathways in multisubunit proteins. Annu Rev Biochem. 1979;48:217–250. doi: 10.1146/annurev.bi.48.070179.001245. [DOI] [PubMed] [Google Scholar]
  8. Garel J. R., Baldwin R. L. Both the fast and slow refolding reactions of ribonuclease A yield native enzyme. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3347–3351. doi: 10.1073/pnas.70.12.3347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goldberg M. E., Creighton T. E., Baldwin R. L., Yanofsky C. Subunit structure of the tryptophan synthetase of Escherichia coli. J Mol Biol. 1966 Oct 28;21(1):71–82. doi: 10.1016/0022-2836(66)90080-5. [DOI] [PubMed] [Google Scholar]
  10. Hathaway G. M., Kida S., Crawford I. P. Subunit structure of the B component of Escherichia coli tryptophan synthetase. Biochemistry. 1969 Mar;8(3):989–997. doi: 10.1021/bi00831a032. [DOI] [PubMed] [Google Scholar]
  11. Jaenicke R. Folding and association of proteins. Biophys Struct Mech. 1982;8(4):231–256. doi: 10.1007/BF00537204. [DOI] [PubMed] [Google Scholar]
  12. Kirschner K., Wiskocil R. L., Foehn M., Rezeau L. The tryptophan synthase from Escherichia coli. An improved purification procedure for the alpha-subunit and binding studies with substrate analogues. Eur J Biochem. 1975 Dec 15;60(2):513–523. doi: 10.1111/j.1432-1033.1975.tb21030.x. [DOI] [PubMed] [Google Scholar]
  13. Labhardt A. M. Secondary structure in ribonuclease. I. Equilibrium folding transitions seen by amide circular dichroism. J Mol Biol. 1982 May 15;157(2):331–355. doi: 10.1016/0022-2836(82)90238-8. [DOI] [PubMed] [Google Scholar]
  14. Lane A. N., Kirschner K. The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes. Eur J Biochem. 1983 Jan 1;129(3):571–582. doi: 10.1111/j.1432-1033.1983.tb07087.x. [DOI] [PubMed] [Google Scholar]
  15. Lane A. N., Kirschner K. The mechanism of binding of L-serine to tryptophan synthase from Escherichia coli. Eur J Biochem. 1983 Jan 1;129(3):561–570. doi: 10.1111/j.1432-1033.1983.tb07086.x. [DOI] [PubMed] [Google Scholar]
  16. Lane A. N., Kirschner K. The mechanism of tryptophan binding to tryptophan synthase from Escherichia coli. Eur J Biochem. 1981 Nov;120(2):379–387. doi: 10.1111/j.1432-1033.1981.tb05715.x. [DOI] [PubMed] [Google Scholar]
  17. Lane A. N., Kirschner K. The quaternary structure of tryptophan synthase from Escherichia coli. Fluorescence and hydrodynamic studies. Eur J Biochem. 1983 Jan 1;129(3):675–684. [PubMed] [Google Scholar]
  18. Lane A. N. The accessibility of the active site and conformation states of the beta 2 subunit of tryptophan synthase studied by fluorescence quenching. Eur J Biochem. 1983 Jul 1;133(3):531–538. doi: 10.1111/j.1432-1033.1983.tb07496.x. [DOI] [PubMed] [Google Scholar]
  19. Lane A. N. The dependence of flux, sensitivity and response of the flux on the concentrations of substrate and modifiers for cooperative enzymes. J Theor Biol. 1982 Dec 7;99(3):491–508. doi: 10.1016/0022-5193(82)90208-9. [DOI] [PubMed] [Google Scholar]
  20. Miles E. W., McPhie P. Evidence for a rate-determining proton abstraction in the serine deaminase reaction of the beta 2 subunit of tryptophan synthetase. J Biol Chem. 1974 May 10;249(9):2852–2857. [PubMed] [Google Scholar]
  21. Miles E. W., Moriguchi M. Tryptophan synthase of Escherichia coli. Removal of pyridoxal 5'-phosphate and separation of the alpha and beta2 subunits. J Biol Chem. 1977 Oct 10;252(19):6594–6599. [PubMed] [Google Scholar]
  22. Miles E. W. The B protein of Escherichia coli tryptophan synthetase. I. Effects of sulfhydryl modification on enzymatic activities and subunit interaction. J Biol Chem. 1970 Nov 25;245(22):6016–6025. [PubMed] [Google Scholar]
  23. Miles E. W. Tryptophan synthase: structure, function, and subunit interaction. Adv Enzymol Relat Areas Mol Biol. 1979;49:127–186. doi: 10.1002/9780470122945.ch4. [DOI] [PubMed] [Google Scholar]
  24. Mosteller R. D., Goldstein R. V., Nishimoto K. R. Interactions of tryptophan synthetase subunits in Escherichia coli containing mutationally altered beta2 subunits. J Biol Chem. 1977 Jul 10;252(13):4527–4532. [PubMed] [Google Scholar]
  25. Paul C., Kirschner K., Haenisch G. Calibration of stopped-flow spectrophotometers using a two-step disulfide exchange reaction. Anal Biochem. 1980 Jan 15;101(2):442–448. doi: 10.1016/0003-2697(80)90211-0. [DOI] [PubMed] [Google Scholar]
  26. Raibaud O., Goldberg M. E. The tryptophanase from Escherichia coli K-12. II. Comparison of the thermal stabilities of apo-, holo-, and hybrid enzymes. J Biol Chem. 1973 May 25;248(10):3451–3455. [PubMed] [Google Scholar]
  27. Tschopp J., Kirschner K. Subunit interactions of tryptophan synthase from Escherichia coli as revealed by binding studies with pyridoxal phosphate analogues. Biochemistry. 1980 Sep 16;19(19):4514–4521. doi: 10.1021/bi00560a020. [DOI] [PubMed] [Google Scholar]
  28. Uehara Y., Tonomura B., Hiromi K. Direct fluorometric determination of a dissociation constant as low as 10(-10) M for the subtilisin BPN'--protein proteinase inhibitor (Streptomyces subtilisin inhibitor) complex by a single photon counting technique. J Biochem. 1978 Nov;84(5):1195–1202. doi: 10.1093/oxfordjournals.jbchem.a132236. [DOI] [PubMed] [Google Scholar]
  29. Welch G. R. On the role of organized multienzyme systems in cellular metabolism: a general synthesis. Prog Biophys Mol Biol. 1977;32(2):103–191. [PubMed] [Google Scholar]
  30. Wiesinger H., Bartholmes P., Hinz H. J. Subunit interaction in tryptophan synthase of Escherichia coli: calorimetric studies on association of alpha and beta 2 subunits. Biochemistry. 1979 May 15;18(10):1979–1984. doi: 10.1021/bi00577a020. [DOI] [PubMed] [Google Scholar]
  31. Wilhelm P., Pilz I., Lane A. N., Kirschner K. Small-angle X-ray scattering studies of tryptophan synthase from Escherichia coli and its alpha and beta 2 subunits. Eur J Biochem. 1982 Dec;129(1):51–56. doi: 10.1111/j.1432-1033.1982.tb07019.x. [DOI] [PubMed] [Google Scholar]
  32. Wilson D. A., Crawford I. P. Purification and properties of the B component of Escherichia coli tryptophan synthetase. J Biol Chem. 1965 Dec;240(12):4801–4808. [PubMed] [Google Scholar]
  33. Yanofsky C., Platt T., Crawford I. P., Nichols B. P., Christie G. E., Horowitz H., VanCleemput M., Wu A. M. The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res. 1981 Dec 21;9(24):6647–6668. doi: 10.1093/nar/9.24.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zetina C. R., Goldberg M. E. Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the beta 2-subunit of Escherichia coli tryptophan-synthetase. J Mol Biol. 1982 May 5;157(1):133–148. doi: 10.1016/0022-2836(82)90516-2. [DOI] [PubMed] [Google Scholar]

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