Mm. Symcox et al., 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 Journal of biological chemistry, 269(37), 1994, pp. 23025-23031
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.