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Volume 271, Number 44, Issue of November 1, 1996 pp. 27630-27636
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Arginine 210 Is Not a Critical Residue for the Allosteric Interactions Mediated by Binding of Cyclic AMP to Site A of Regulatory (RI) Subunit of Cyclic AMP-dependent Protein Kinase

(Received for publication, May 7, 1996, and in revised form, July 29, 1996)

Robert A. Steinberg , Marina M. Symcox , Snorre Sollid and Dagfinn Øgreid

From the  Department of Biochemistry and Molecular Biology, the University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 and the  Center of Molecular Medicine, University of Bergen, Haukeland Hospital, N-5021 Bergen, Norway

The guanidinium groups of conserved arginines in the two intrachain cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase have been implicated in the allosteric interactions by which cAMP binding leads to kinase activation. We have investigated the functional role of Arg-210, the conserved arginine in site A of murine type I R subunit, by analyzing the effects of nine different substitutions at this residue on cAMP binding and allosteric properties of bacterially expressed RI subunits. All substitutions reduced the cAMP binding affinity of site A, but the magnitude of reduction varied from several hundredfold to 10⁶-fold. The differential effects of the different substitutions could not easily be rationalized by interactions with cAMP and might, in part, reflect interactions with other residues in the unoccupied cAMP-binding pocket. None of the Arg-210 substitutions appeared to disrupt the allosteric interaction by which occupation of site A slows dissociation of cAMP from site B, although the effect was difficult to elicit in full with mutations that had strong effects on cAMP binding. The two weakest substitutions, Arg-210  Ile and Arg-210  Thr, could be shown to have essentially no effect on the allosteric interaction by which occupation of site A reduces the affinity of R subunit for the catalytic subunit. The weaker mutations had a smaller effect on kinase activation by the suboptimal activator R_p-adenosine cyclic 3,5-phosphorothioate than by cAMP, suggesting that the analog largely bypasses interactions with the guanidinium group of Arg-210.

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