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. 2000 Sep;9(9):1818–1827. doi: 10.1110/ps.9.9.1818

Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives.

Q Ni 1, J Shaffer 1, J A Adams 1
PMCID: PMC2144695  PMID: 11045627

Abstract

The binding of the methylanthraniloyl derivatives of ATP (mant-ATP), ADP (mant-ADP), 2'deoxyATP (mant-2'deoxyATP), and 3'deoxyATP (mant-3'deoxyATP) to the catalytic subunit of protein kinase A was studied to gain insights into the mechanism of nucleotide binding. The binding of the mant nucleotides leads to a large increase in fluorescence energy transfer at 440 nm, allowing direct measurements of nucleotide affinity. The dissociation constant of mant-ADP is identical to that for ADP, while that for mant-ATP is approximately threefold higher than that for ATP. The dissociation constant for mant-3'deoxyATP is approximately fivefold higher than that for 3'deoxyATP while derivatization of 2'deoxyATP does not affect affinity. The time-dependent binding of mant-ATP, mant-2'deoxyATP, and mant-ADP, measured using stopped-flow fluorescence spectroscopy, is best fit to three exponentials. The fast phase is ligand dependent, while the two slower phases are ligand independent. The slower phases are similar but not identical in rate, and have opposite fluorescence amplitudes. Both isomers of mant-ATP are equivalent substrates, as judged by reversed-phase chromatography, although the rate of phosphorylation is approximately 20-fold lower than the natural nucleotide. The kinetic data are consistent with a three-step binding mechanism in which initial association of the nucleotide derivatives produces a highly fluorescent complex. Either one or two conformational changes can occur after the formation of this binary species, but one of the isomerized forms must have low fluorescence compared to the initial binary complex. These data soundly attest to the structural plasticity within the kinase core that may be essential for catalysis. Overall, the mant nucleotides present a useful reporter system for gauging these conformational changes in light of the prevailing three-dimensional models for the enzyme.

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Selected References

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  1. Adams J. A., Taylor S. S. Divalent metal ions influence catalysis and active-site accessibility in the cAMP-dependent protein kinase. Protein Sci. 1993 Dec;2(12):2177–2186. doi: 10.1002/pro.5560021217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams J. A., Taylor S. S. Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments. Biochemistry. 1992 Sep 15;31(36):8516–8522. doi: 10.1021/bi00151a019. [DOI] [PubMed] [Google Scholar]
  3. Armstrong R. N., Kondo H., Granot J., Kaiser E. T., Mildvan A. S. Magnetic resonance and kinetic studies of the manganese(II) ion and substrate complexes of the catalytic subunit of adenosine 3',5'-monophosphate dependent protein kinase from bovine heart. Biochemistry. 1979 Apr 3;18(7):1230–1238. doi: 10.1021/bi00574a018. [DOI] [PubMed] [Google Scholar]
  4. Armstrong R. N., Kondo H., Kaiser E. T. Cyclic AMP-dependent ATPase activity of bovine heart protein kinase. Proc Natl Acad Sci U S A. 1979 Feb;76(2):722–725. doi: 10.1073/pnas.76.2.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bhatnagar D., Roskoski R., Jr, Rosendahl M. S., Leonard N. J. Adenosine cyclic 3',5'-monophosphate dependent protein kinase: a new fluorescence displacement titration technique for characterizing the nucleotide binding site on the catalytic subunit. Biochemistry. 1983 Dec 20;22(26):6310–6317. doi: 10.1021/bi00295a042. [DOI] [PubMed] [Google Scholar]
  6. Chan A. C., Kadlecek T. A., Elder M. E., Filipovich A. H., Kuo W. L., Iwashima M., Parslow T. G., Weiss A. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science. 1994 Jun 10;264(5165):1599–1601. doi: 10.1126/science.8202713. [DOI] [PubMed] [Google Scholar]
  7. Cheng J. Q., Jiang W., Hackney D. D. Interaction of mant-adenosine nucleotides and magnesium with kinesin. Biochemistry. 1998 Apr 14;37(15):5288–5295. doi: 10.1021/bi972742j. [DOI] [PubMed] [Google Scholar]
  8. Cheng X., Shaltiel S., Taylor S. S. Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting. Biochemistry. 1998 Oct 6;37(40):14005–14013. doi: 10.1021/bi981057p. [DOI] [PubMed] [Google Scholar]
  9. Clauser E., Leconte I., Auzan C. Molecular basis of insulin resistance. Horm Res. 1992;38(1-2):5–12. doi: 10.1159/000182470. [DOI] [PubMed] [Google Scholar]
  10. Cohen P. The development and therapeutic potential of protein kinase inhibitors. Curr Opin Chem Biol. 1999 Aug;3(4):459–465. doi: 10.1016/S1367-5931(99)80067-2. [DOI] [PubMed] [Google Scholar]
  11. Cook P. F., Neville M. E., Jr, Vrana K. E., Hartl F. T., Roskoski R., Jr Adenosine cyclic 3',5'-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit. Biochemistry. 1982 Nov 9;21(23):5794–5799. doi: 10.1021/bi00266a011. [DOI] [PubMed] [Google Scholar]
  12. Elder M. E., Lin D., Clever J., Chan A. C., Hope T. J., Weiss A., Parslow T. G. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science. 1994 Jun 10;264(5165):1596–1599. doi: 10.1126/science.8202712. [DOI] [PubMed] [Google Scholar]
  13. Fierke C. A., Hammes G. G. Transient kinetic approaches to enzyme mechanisms. Methods Enzymol. 1995;249:3–37. doi: 10.1016/0076-6879(95)49029-9. [DOI] [PubMed] [Google Scholar]
  14. Friedman A. L., Geeves M. A., Manstein D. J., Spudich J. A. Kinetic characterization of myosin head fragments with long-lived myosin.ATP states. Biochemistry. 1998 Jul 7;37(27):9679–9687. doi: 10.1021/bi973143f. [DOI] [PubMed] [Google Scholar]
  15. Grant B. D., Adams J. A. Pre-steady-state kinetic analysis of cAMP-dependent protein kinase using rapid quench flow techniques. Biochemistry. 1996 Feb 13;35(6):2022–2029. doi: 10.1021/bi952144+. [DOI] [PubMed] [Google Scholar]
  16. Hartl F. T., Roskoski R., Jr, Rosendahl M. S., Leonard N. J. Adenosine cyclic 3',5'-monophosphate dependent protein kinase: interaction of the catalytic subunit and holoenzyme with lin-benzoadenine nucleotides. Biochemistry. 1983 May 10;22(10):2347–2352. doi: 10.1021/bi00279a007. [DOI] [PubMed] [Google Scholar]
  17. Hiratsuka T. New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. Biochim Biophys Acta. 1983 Feb 15;742(3):496–508. doi: 10.1016/0167-4838(83)90267-4. [DOI] [PubMed] [Google Scholar]
  18. Hoppe J., Freist W., Marutzky R., Shaltiel S. Mapping the ATP-binding site in the catalytic subunit of adenosine-3':5'-monophosphate-dependent protein kinase. Spatial relationship with the ATP site of the undissociated enzyme. Eur J Biochem. 1978 Oct 16;90(3):427–432. doi: 10.1111/j.1432-1033.1978.tb12621.x. [DOI] [PubMed] [Google Scholar]
  19. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  20. Knighton D. R., Zheng J. H., Ten Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. doi: 10.1126/science.1862343. [DOI] [PubMed] [Google Scholar]
  21. Kong C. T., Cook P. F. Isotope partitioning in the adenosine 3',5'-monophosphate dependent protein kinase reaction indicates a steady-state random kinetic mechanism. Biochemistry. 1988 Jun 28;27(13):4795–4799. doi: 10.1021/bi00413a032. [DOI] [PubMed] [Google Scholar]
  22. Lew J., Taylor S. S., Adams J. A. Identification of a partially rate-determining step in the catalytic mechanism of cAMP-dependent protein kinase: a transient kinetic study using stopped-flow fluorescence spectroscopy. Biochemistry. 1997 Jun 3;36(22):6717–6724. doi: 10.1021/bi963164u. [DOI] [PubMed] [Google Scholar]
  23. Liu X., Pawson T. Biochemistry of the Src protein-tyrosine kinase: regulation by SH2 and SH3 domains. Recent Prog Horm Res. 1994;49:149–160. doi: 10.1016/b978-0-12-571149-4.50011-8. [DOI] [PubMed] [Google Scholar]
  24. Ma Y. Z., Taylor E. W. Kinetic mechanism of a monomeric kinesin construct. J Biol Chem. 1997 Jan 10;272(2):717–723. doi: 10.1074/jbc.272.2.717. [DOI] [PubMed] [Google Scholar]
  25. Madhusudan, Trafny E. A., Xuong N. H., Adams J. A., Ten Eyck L. F., Taylor S. S., Sowadski J. M. cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer. Protein Sci. 1994 Feb;3(2):176–187. doi: 10.1002/pro.5560030203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mitchell R. D., Glass D. B., Wong C. W., Angelos K. L., Walsh D. A. Heat-stable inhibitor protein derived peptide substrate analogs: phosphorylation by cAMP-dependent and cGMP-dependent protein kinases. Biochemistry. 1995 Jan 17;34(2):528–534. doi: 10.1021/bi00002a018. [DOI] [PubMed] [Google Scholar]
  27. Mocz G., Helms M. K., Jameson D. M., Gibbons I. R. Probing the nucleotide binding sites of axonemal dynein with the fluorescent nucleotide analogue 2'(3')-O-(-N-Methylanthraniloyl)-adenosine 5'-triphosphate. Biochemistry. 1998 Jul 7;37(27):9862–9869. doi: 10.1021/bi9730184. [DOI] [PubMed] [Google Scholar]
  28. Moyer M. L., Gilbert S. P., Johnson K. A. Pathway of ATP hydrolysis by monomeric and dimeric kinesin. Biochemistry. 1998 Jan 20;37(3):800–813. doi: 10.1021/bi9711184. [DOI] [PubMed] [Google Scholar]
  29. Narayana N., Cox S., Nguyen-huu X., Ten Eyck L. F., Taylor S. S. A binary complex of the catalytic subunit of cAMP-dependent protein kinase and adenosine further defines conformational flexibility. Structure. 1997 Jul 15;5(7):921–935. doi: 10.1016/s0969-2126(97)00246-3. [DOI] [PubMed] [Google Scholar]
  30. Olah G. A., Mitchell R. D., Sosnick T. R., Walsh D. A., Trewhella J. Solution structure of the cAMP-dependent protein kinase catalytic subunit and its contraction upon binding the protein kinase inhibitor peptide. Biochemistry. 1993 Apr 13;32(14):3649–3657. doi: 10.1021/bi00065a018. [DOI] [PubMed] [Google Scholar]
  31. Pawson T., Scott J. D. Signaling through scaffold, anchoring, and adaptor proteins. Science. 1997 Dec 19;278(5346):2075–2080. doi: 10.1126/science.278.5346.2075. [DOI] [PubMed] [Google Scholar]
  32. Rawlings D. J., Saffran D. C., Tsukada S., Largaespada D. A., Grimaldi J. C., Cohen L., Mohr R. N., Bazan J. F., Howard M., Copeland N. G. Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice. Science. 1993 Jul 16;261(5119):358–361. doi: 10.1126/science.8332901. [DOI] [PubMed] [Google Scholar]
  33. Shaffer J., Adams J. A. An ATP-linked structural change in protein kinase A precedes phosphoryl transfer under physiological magnesium concentrations. Biochemistry. 1999 Apr 27;38(17):5572–5581. doi: 10.1021/bi982768q. [DOI] [PubMed] [Google Scholar]
  34. Shaffer J., Adams J. A. Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique. Biochemistry. 1999 Sep 14;38(37):12072–12079. doi: 10.1021/bi991109q. [DOI] [PubMed] [Google Scholar]
  35. Thomas J. D., Sideras P., Smith C. I., Vorechovský I., Chapman V., Paul W. E. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science. 1993 Jul 16;261(5119):355–358. doi: 10.1126/science.8332900. [DOI] [PubMed] [Google Scholar]
  36. Török K., Trentham D. R. Mechanism of 2-chloro-(epsilon-amino-Lys75)-[6-[4-(N,N- diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin interactions with smooth muscle myosin light chain kinase and derived peptides. Biochemistry. 1994 Nov 1;33(43):12807–12820. doi: 10.1021/bi00209a012. [DOI] [PubMed] [Google Scholar]
  37. Woodward S. K., Eccleston J. F., Geeves M. A. Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes. Biochemistry. 1991 Jan 15;30(2):422–430. doi: 10.1021/bi00216a017. [DOI] [PubMed] [Google Scholar]
  38. Yonemoto W. M., McGlone M. L., Slice L. W., Taylor S. S. Prokaryotic expression of catalytic subunit of adenosine cyclic monophosphate-dependent protein kinase. Methods Enzymol. 1991;200:581–596. doi: 10.1016/0076-6879(91)00173-t. [DOI] [PubMed] [Google Scholar]
  39. Zheng J., Knighton D. R., Xuong N. H., Taylor S. S., Sowadski J. M., Ten Eyck L. F. Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations. Protein Sci. 1993 Oct;2(10):1559–1573. doi: 10.1002/pro.5560021003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zheng J., Knighton D. R., ten Eyck L. F., Karlsson R., Xuong N., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry. 1993 Mar 9;32(9):2154–2161. doi: 10.1021/bi00060a005. [DOI] [PubMed] [Google Scholar]
  41. Zhou J., Adams J. A. Participation of ADP dissociation in the rate-determining step in cAMP-dependent protein kinase. Biochemistry. 1997 Dec 16;36(50):15733–15738. doi: 10.1021/bi971438n. [DOI] [PubMed] [Google Scholar]

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