ARGININE-210 IS NOT A CRITICAL RESIDUE FOR THE ALLOSTERIC INTERACTIONS MEDIATED BY BINDING OF CYCLIC-AMP TO SITE-A OF REGULATORY (RI-ALPHA)SUBUNIT OF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE

The guanidinium groups of conserved arginines in the two intrachain cA MP-binding sites of regulatory (R) subunit of cAMP-dependent protein k inase have been implicated in the allosteric interactions by which cAM P binding leads to kinase activation, We have investigated the functio nal role of Arg-210, the conserved arginine in site A of murine type I alpha R subunit, by analyzing the effects of nine different substitut ions at this residue on cAMP binding and allosteric properties of bact erially expressed RI alpha subunits, All substitutions reduced the cAM P binding affinity of site A, but the magnitude of reduction varied fr om several hundredfold to 10(6)-fold, The differential effects of the different substitutions could not easily be rationalized by interactio ns with cAMP and might, in part, reflect interactions with other resid ues in She unoccupied cAMP-binding pocket, None of the Arg-210 substit utions appeared to disrupt the allosteric interaction by which occupat ion of site a slows dissociation of cAMP from site B, although the eff ect was difficult to elicit in full with mutations that had strong eff ects on cAMP binding, The two weakest substitutions, Arg-210 --> Ile a nd Arg-210 --> Thr, could be shown to have: essentially no effect on t he allosteric interaction by which occupation of site A reduces the af finity of R subunit for the catalytic subunit, The weaker mutations ha d a smaller effect on kinase activation by the suboptimal activator R( p)-adenosine cyclic 3',5'-phosphorothioate than by cAMP, suggesting th at the analog largely bypasses interactions with the guanidinium group of Arg-210.