Arg-242 is necessary for allosteric coupling of cyclic AMP-binding sites A and 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 Escherichia coli. These mutations arose in cAMP-resistant mutants to S49 mouse lymphoma cells and were shown previously to inhibit cAMP binding to site A, the more amino-terminal of two intrachain cAMP-binding sites. Binding of cAMP to site A of the mutant R subunits could be detected by cAMP-dependent quenching of endogenous tryptophan fluorescence, [3H]cAMP binding to mutant R subunits with the Arg-242 mutations without or with an inactivating mutation in site B, or biphasic dissociation of [3H]cAMP from the mutant subunits at low temperature. The mutations reduced site A affinities by about 25-fold, and the reductions were attributable to accelerated rates of cAMP dissociation. While the presence of cAMP in site A retards dissociation of [3H]cAMP from site B of wild-type R subunits, saturation of site A had little or no effect on dissociation of [3H]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 interaction, that coupling occupation of site A with a reduced affinity of R for catalytic subunit, was inhibited only partially by these mutations at Arg-242.
Symcox
M M
MM
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Cauthron
R D
RD
Ogreid
D
D
Steinberg
R A
RA
D000494:Allosteric Regulation; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D014407:Tumor Cells, Cultured
D001120:Arginine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases