Allosteric cooperativity between peptide and ATP binding sites on cAMP-dependent protein kinase catalytic subunit was studied kinetically for the reaction of phosphorylation of seven peptide substrates. The allosteric effect was quantified in terms of the interaction factor by comparing binding effectiveness of a substrate molecule with the free enzyme and with the enzyme complex with another substrate. It was discovered that the magnitude of the allosteric feedback between these binding sites was governed by the effectiveness of substrate binding, which was varied by using different peptides, and the principle ‘better binding: stronger allostery’ was formulated. This interrelationship was further formalized in terms of a linear-free-energy relationship pα = C + SpKb, holding between the free energy of the allosteric interaction, quantified by the negative logarithm of the interaction factor α (pα), and the effectiveness of substrate binding quantified by pKb For the peptide phosphorylation reaction C = -1.4 and S = 0.4 were obtained. The negative intercept indicated that the positive cooperativity between the binding sites, characterized by α < 1 at sub-millimolar Kb values, changed into negative cooperativity with α > 1 at millimolar Kb values. This means that inversion of the cooperative effect was induced by substrate structure, and allostery was used by this enzyme as an additional mechanism to discriminate between substrates, facilitating phosphorylation of good substrates and providing additional protection against phosphorylation of bad substrates. Some implications of this allosteric mechanism on substrate specificity of protein kinases were discussed.
1. Monod, J., Wyman, J., and Changeux, J. P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol., 1965, 12, 88–118.
2. Koshland, D. E., Jr. and Hamadani, K. Proteomics and models for enzyme cooperativity. J. Biol. Chem., 2002, 277, 46841–46844.
doi:10.1074/jbc.R200014200
3. Lindsley, J. E. and Rutter, J. Whence cometh the allosterome? Proc. Natl. Acad. Sci. USA, 2006, 103, 10533–10535.
doi:10.1073/pnas.0604452103
4. Gunasekaran, K., Ma, B., and Nussinov, R. Is allostery an intrinsic property of all dynamic proteins? Proteins, 2004, 57, 433–443.
doi:10.1002/prot.20232
5. Hanks, S. K. and Hunter, T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. Faseb J., 1995, 9, 576–596.
6. Taylor, S. S. cAMP-dependent protein kinase. Model for an enzyme family. J. Biol. Chem., 1989, 264, 8443–8446.
7. Graves, J. D. and Krebs, E. G. Protein phosphorylation and signal transduction. Pharmacol. Ther., 1999, 82, 111–121.
doi:10.1016/S0163-7258(98)00056-4
8. Manning, G., Plowman, G. D., Hunter, T., and Sudarsanam, S. Evolution of protein kinase signaling from yeast to man. Trends Biochem. Sci., 2002, 27, 514–520.
doi:10.1016/S0968-0004(02)02179-5
9. Ho, M., Bramson, H. N., Hansen, D. E., Knowles, J. R., et al. Stereochemical course of the phospho group transfer catalyzed by cAMP-dependent protein kinase. J. Am. Chem. Soc., 1988, 110, 2680–2681.
doi:10.1021/ja00216a068
10. Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems. Wiley, New York, 1975.
11. Herberg, F. W. and Taylor, S. S. Physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase: effect of MgATP on protein–protein interactions. Biochemistry, 1993, 32, 14015–14022.
doi:10.1021/bi00213a035
12. Whitehouse, S., Feramisco, J. R., Casnellie, J. E., Krebs, E. G., et al. Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase. J. Biol. Chem., 1983, 258, 3693–3701.
13. Masterson, L. R., Mascioni, A., Traaseth, N. J., Taylor, S. S., et al. Allosteric cooperativity in protein kinase A. Proc. Natl. Acad. Sci. USA, 2008, 105, 506–511.
doi:10.1073/pnas.0709214104
14. Kuznetsov, A. and Järv, J. Allosteric cooperativity in inhibition of protein kinase A catalytic subunit. Open Enzyme Inhib. J., 2008, 1, 42–47.
doi:10.2174/1874940200801010042
15. Roskoski, R., Jr. Assays of protein kinase. Methods Enzymol., 1983, 99, 3–6.
doi:10.1016/0076-6879(83)99034-1
16. Kuznetsov, A., Uri, A., Raidaru, G., and Järv, J. Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide-nucleoside conjugate AdcAhxArg6. Bioorg. Chem., 2004, 32, 527–535.
doi:10.1016/j.bioorg.2004.05.004
17. Kuznetsov, A., Väärtnõu-Järv, H., and Järv, J. Kinetic model for protein kinase simultaneous interaction with peptide, ATP and bifunctional inhibitor. Proc. Estonian Acad. Sci. Chem., 2003, 52, 178–187.
18. Symcox, M. M. and Reinhart, G. D. A steady-state kinetic method for the verification of the rapid-equilibrium assumption in allosteric enzymes. Anal. Biochem., 1992, 206, 394–399.
doi:10.1016/0003-2697(92)90384-J
19. Glass, D. B. and Krebs, E. G. Comparison of the substrate specificity of adenosine 3¢ : 5¢-monophosphate- and guanosine 3¢ : 5¢-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B. J. Biol. Chem., 1979, 254, 9728–9738.
20. Wu, J., Ma, Q. N., and Lam, K. S. Identifying substrate motifs of protein kinases by a random library approach. Biochemistry, 1994, 33, 14825–14833.
doi:10.1021/bi00253a022
21. Hider, R. C., Ragnarsson, U., and Zetterqvist, O. The role of the phosphate group for the structure of phosphopeptide products of adenosine 3¢,5¢-cyclic monophosphate-dependent protein kinase. Biochem. J., 1985, 229, 485–489.
22. Kemp, B. E., Graves, D. J., Benjamini, E., and Krebs, E. G. Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J. Biol. Chem., 1977, 252, 4888–4894.
23. Daile, P., Carnegie, P. R., and Young, J. D. Synthetic substrate for cyclic AMP-dependent protein kinase. Nature, 1975, 257, 416–418.
doi:10.1038/257416a0
24. Knowles, J. R. Enzyme specificity: alpha-chymotrypsin. J. Theor. Biol., 1965, 9, 213–228.
doi:10.1016/0022-5193(65)90108-6
25. Järv, J., Kesvatera, T., and Aavisksaar, A. Structure–activity relationships in acetylcholinesterase reactions. Hydrolysis of non-ionic acetic esters. Eur. J. Biochem., 1976, 67, 315–322.
doi:10.1111/j.1432-1033.1976.tb10694.x
26. Järv, J. L. and Langel, Ü. L. Substrate leaving group effects in the butyrylcholinesterase catalyzed reactions.Bioorg. Khim., 1979, 5, 746–756 (in Russian).
27. Tsai, C. J., del Sol, A., and Nussinov, R. Allostery: absence of a change in shape does not imply that allostery is not at play. J. Mol. Biol., 2008, 378, 1–11.
doi:10.1016/j.jmb.2008.02.034
28. Chi, C. N., Elfström, L., Shi, Y., Snäll, T., Engström, Å., and Jemth, P. Reassessing a sparse energetic network within a single protein domain. Proc. Natl. Acad. Sci. USA, 2008, 105, 4679–4684.
doi:10.1073/pnas.0711732105
