ARG-242 IS NECESSARY FOR ALLOSTERIC COUPLING OF CYCLIC AMP-BINDING SITE-A AND SITE-B OF RI-SUBUNIT OF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE

The functional consequences of Arg-242 to Ser or Lys substitutions in type I alpha regulatory (R) subunits of cAMP-dependent protein kinase were analyzed by using recombinant murine R subunits expressed in Esch erichia coli. These mutations arose in cAMP-resistant mutants to S49 m ouse lymphoma cells and were shown previously to inhibit cAMP binding to site A, the more amino-terminal of two intrachain cAMP-binding site s. Binding of cAMP to site A of the mutant R subunits could be detecte d by cAMP-dependent quenching of endogenous tryptophan fluorescence, [ H-3]cAMP binding to mutant R subunits with the Arg-242 mutations witho ut or with an inactivating mutation in site B, or biphasic dissociatio n of [H-3]cAMP from the mutant subunits at low temperature. The mutati ons reduced site A affinities by about 25-fold, and the reductions wer e attributable to accelerated rates of cAMP dissociation. While the pr esence of cAMP in site A retards dissociation of [H-3]cAMP from site B of wild-type R subunits, saturation of site A had little or no effect on dissociation of [H-3]cAMP from site B of the mutant subunits. The predominant effect of the mutations, therefore, was loss of allosteric coupling between the two cAMP-binding sites. A second allosteric inte raction, that coupling occupation of site A with a reduced affinity of R for catalytic subunit, was inhibited only partially by these mutati ons at Arg-242.