Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1980 Sep 11;8(17):4021–4039. doi: 10.1093/nar/8.17.4021

Serine activation is the rate limiting step of tRNASer aminoacylation by yeast seryl tRNA synthetase.

L Dibbelt, U Pachmann, H G Zachau
PMCID: PMC324212  PMID: 6777760

Abstract

Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.

Full text

PDF
4021

Selected References

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

  1. Arnott S., Hukins D. W. Refinement of the structure of B-DNA and implications for the analysis of x-ray diffraction data from fibers of biopolymers. J Mol Biol. 1973 Dec 5;81(2):93–105. doi: 10.1016/0022-2836(73)90182-4. [DOI] [PubMed] [Google Scholar]
  2. Blanquet S., Dessen P. Antico-operative binding of bacterial and mammalian initiator tRNAMet to methionyl-tRNA synthetase from escherichia coli. J Mol Biol. 1976 Jun 5;103(4):765–784. doi: 10.1016/0022-2836(76)90208-4. [DOI] [PubMed] [Google Scholar]
  3. Corden J., Engelking H. M., Pearson G. D. Chromatin-like organization of the adenovirus chromosome. Proc Natl Acad Sci U S A. 1976 Feb;73(2):401–404. doi: 10.1073/pnas.73.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Crick F. H., Klug A. Kinky helix. Nature. 1975 Jun 12;255(5509):530–533. doi: 10.1038/255530a0. [DOI] [PubMed] [Google Scholar]
  5. Deininger P., Esty A., LaPorte P., Friedmann T. Nucleotide sequence and genetic organization of the polyoma late region: features common to the polyoma early region and SV40. Cell. 1979 Nov;18(3):771–779. doi: 10.1016/0092-8674(79)90130-2. [DOI] [PubMed] [Google Scholar]
  6. Fasiolo F., Fersht A. R. The aminoacyladenylate mechanism in the aminoacylation reaction of yeast phenylalanyl-tRNA synthetase. Eur J Biochem. 1978 Apr;85(1):85–88. doi: 10.1111/j.1432-1033.1978.tb12214.x. [DOI] [PubMed] [Google Scholar]
  7. Feigon J., Kearns D. R. 1H NMR investigation of the conformational states of DNA in nucleosome core particles. Nucleic Acids Res. 1979;6(6):2327–2337. doi: 10.1093/nar/6.6.2327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fersht A. R., Gangloff J., Dirheimer G. Reaction pathway and rate-determining step in the aminoacylation of tRNAArg catalyzed by the arginyl-tRNA synthetase from yeast. Biochemistry. 1978 Sep 5;17(18):3740–3746. doi: 10.1021/bi00611a011. [DOI] [PubMed] [Google Scholar]
  9. Fersht A. R., Jakes R. Demonstration of two reaction pathways for the aminoacylation of tRNA. Application of the pulsed quenched flow technique. Biochemistry. 1975 Jul 29;14(15):3350–3356. doi: 10.1021/bi00686a010. [DOI] [PubMed] [Google Scholar]
  10. Fersht A. R., Kaethner M. M. Mechanism of aminoacylation of tRNA. Proof of the aminoacyl adenylate pathway for the isoleucyl- and tyrosyl-tRNA synthetases from Escherichia coli K12. Biochemistry. 1976 Feb 24;15(4):818–823. doi: 10.1021/bi00649a014. [DOI] [PubMed] [Google Scholar]
  11. Fiers W., Contreras R., Haegemann G., Rogiers R., Van de Voorde A., Van Heuverswyn H., Van Herreweghe J., Volckaert G., Ysebaert M. Complete nucleotide sequence of SV40 DNA. Nature. 1978 May 11;273(5658):113–120. doi: 10.1038/273113a0. [DOI] [PubMed] [Google Scholar]
  12. Friedmann T., Esty A., LaPorte P., Deininger P. The nucleotide sequence and genome organization of the polyoma early region: extensive nucleotide and amino acid homology with SV40. Cell. 1979 Jul;17(3):715–724. doi: 10.1016/0092-8674(79)90278-2. [DOI] [PubMed] [Google Scholar]
  13. Germond J. E., Hirt B., Oudet P., Gross-Bellark M., Chambon P. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A. 1975 May;72(5):1843–1847. doi: 10.1073/pnas.72.5.1843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hirsch R., Zachau H. G. Isolierung und Charakterisierung der Seryl- und Phenylalanyl-tRNA-Synthetase aus Hefe. Hoppe Seylers Z Physiol Chem. 1976 Apr;357(4):509–526. [PubMed] [Google Scholar]
  15. Kallenbach N. R., Appleby D. W., Bradley C. H. 31P magnetic resonance of DNA in nucleosome core particles of chromatin. Nature. 1978 Mar 9;272(5649):134–138. doi: 10.1038/272134a0. [DOI] [PubMed] [Google Scholar]
  16. Kröger A., Klingenberg M. The kinetics of the redox reactions of ubiquinone related to the electron-transport activity in the respiratory chain. Eur J Biochem. 1973 Apr;34(2):358–368. doi: 10.1111/j.1432-1033.1973.tb02767.x. [DOI] [PubMed] [Google Scholar]
  17. Lagerkvist U., Akesson B., Brändén R. Aminoacyl adenylate, a normal intermediate or a dead end in aminoacylation of transfer ribonucleic acid. J Biol Chem. 1977 Feb 10;252(3):1002–1006. [PubMed] [Google Scholar]
  18. Levitt M. How many base-pairs per turn does DNA have in solution and in chromatin? Some theoretical calculations. Proc Natl Acad Sci U S A. 1978 Feb;75(2):640–644. doi: 10.1073/pnas.75.2.640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maat J., Van Ormondt H. The nucleotide sequence of the transforming HindIII-G fragment of adenovirus type 5 DNA. The region between map positions 4.5 (HpaI site) and 8.0 (HindIII site). Gene. 1979 May;6(1):75–90. doi: 10.1016/0378-1119(79)90086-6. [DOI] [PubMed] [Google Scholar]
  20. Mulvey R. S., Fersht A. R. Mechanism of aminoacylation of transfer RNA. A pre-steady-state analysis of the reaction pathway catalyzed by the methionyl-tRNA synthetase of Bacillus stearothermophilus. Biochemistry. 1978 Dec 26;17(26):5591–5597. doi: 10.1021/bi00619a002. [DOI] [PubMed] [Google Scholar]
  21. Namoradze N. Z., Goryunov A. N., Birshtein T. M. On conformations of the superhelix structure. Biophys Chem. 1977 Jun;7(1):59–70. doi: 10.1016/0301-4622(77)87015-4. [DOI] [PubMed] [Google Scholar]
  22. Pachmann U., Zachau H. G. Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation. Nucleic Acids Res. 1978 Mar;5(3):961–973. doi: 10.1093/nar/5.3.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reddy V. B., Thimmappaya B., Dhar R., Subramanian K. N., Zain B. S., Pan J., Ghosh P. K., Celma M. L., Weissman S. M. The genome of simian virus 40. Science. 1978 May 5;200(4341):494–502. doi: 10.1126/science.205947. [DOI] [PubMed] [Google Scholar]
  24. Riesner D., Pingoud A., Boehme D., Peters F., Maass G. Distinct steps in the specific binding of tRNA to aminoacyl-tRNA synthetase. Temperature-jump studies on the serine-specific system from yeast and the tyrosine-specific system from Escherichia coli. Eur J Biochem. 1976 Sep;68(1):71–80. doi: 10.1111/j.1432-1033.1976.tb10765.x. [DOI] [PubMed] [Google Scholar]
  25. Rigler R., Pachmann U., Hirsch R., Zachau H. G. On the interaction of seryl-tRNA synthetase with tRNA Ser. A contribution to the problem of synthetase-tRNA recognition. Eur J Biochem. 1976 May 17;65(1):307–315. doi: 10.1111/j.1432-1033.1976.tb10418.x. [DOI] [PubMed] [Google Scholar]
  26. Robertson M. A., Staden R., Tanaka Y., Catterall J. F., O'Malley B. W., Brownlee G. G. Sequence of three introns in the chick ovalbumin gene. Nature. 1979 Mar 22;278(5702):370–372. doi: 10.1038/278370a0. [DOI] [PubMed] [Google Scholar]
  27. Sobell H. M., Tsai C. C., Gilbert S. G., Jain S. C., Sakore T. D. Organization of DNA in chromatin. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3068–3072. doi: 10.1073/pnas.73.9.3068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sures I., Lowry J., Kedes L. H. The DNA sequence of sea urchin (S. purpuratus) H2A, H2B and H3 histone coding and spacer regions. Cell. 1978 Nov;15(3):1033–1044. doi: 10.1016/0092-8674(78)90287-8. [DOI] [PubMed] [Google Scholar]
  29. Sussman J. L., Trifonov E. N. Possibility of nonkinked packing of DNA in chromatin. Proc Natl Acad Sci U S A. 1978 Jan;75(1):103–107. doi: 10.1073/pnas.75.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thiebe R., Hirsch R. A test for the active intermediate in the aminoacylation of tRNAPhe from yeast. FEBS Lett. 1975 Dec 15;60(2):338–341. doi: 10.1016/0014-5793(75)80744-7. [DOI] [PubMed] [Google Scholar]
  31. Trifonov E. N., Bettecken T. Noninteger pitch and nuclease sensitivity of chromatin DNA. Biochemistry. 1979 Feb 6;18(3):454–456. doi: 10.1021/bi00570a011. [DOI] [PubMed] [Google Scholar]
  32. Tsujimoto Y., Suzuki Y. The DNA sequence of Bombyx mori fibroin gene including the 5' flanking, mRNA coding, entire intervening and fibroin protein coding regions. Cell. 1979 Oct;18(2):591–600. doi: 10.1016/0092-8674(79)90075-8. [DOI] [PubMed] [Google Scholar]
  33. Van Ormondt H., Maat J., De Waard A., Van der Eb A. J. The nucleotide sequence of the transforming HpaI-E fragment of adenovirus type 5 DNA. Gene. 1978 Dec;4(4):309–328. doi: 10.1016/0378-1119(78)90048-3. [DOI] [PubMed] [Google Scholar]
  34. Zachau H. G., Hertz H. S. Lack of incorporation of 1:N6-ethenoadenosine triphosphate into yeast tRNAPhe and tRNASer under standard conditions. Eur J Biochem. 1974 May 2;44(1):289–291. doi: 10.1111/j.1432-1033.1974.tb03484.x. [DOI] [PubMed] [Google Scholar]
  35. Zhurkin V. B., Lysov Y. P., Ivanov V. I. Anisotropic flexibility of DNA and the nucleosomal structure. Nucleic Acids Res. 1979 Mar;6(3):1081–1096. doi: 10.1093/nar/6.3.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES