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J. Biol. Chem., Vol. 265, Issue 9, 4800-4808, Mar, 1990
GE Ringheim and SS Taylor
A truncated regulatory subunit of cAMP-dependent protein kinase I was
constructed which contained deletions at both the carboxyl terminus and at
the amino terminus. The entire carboxyl-terminal cAMP-binding domain was
deleted as well as the first 92 residues up to the hinge region. This
monomeric truncated protein still forms a complex with the catalytic
subunit, and activation of this complex is mediated by cAMP. The affinity
of this mutant holoenzyme for cAMP and its activation by cAMP are nearly
identical to holoenzyme formed with a regulatory subunit having only the
carboxyl-terminal deletion and very similar to native holoenzyme. The off
rate for cAMP from both mutant regulatory subunits, however, is monophasic
and very fast relative to the biphasic off rate seen for the native
regulatory subunit. The effects of NaCl, urea, and pH on cAMP binding are
also very similar for the mutant and native holoenzymes. Like the native
type I holoenzyme, both mutant holoenzymes bind ATP with a high affinity.
The positive cooperativity seen for MgATP binding to the native holoenzyme,
however, is abolished in the double deletion mutant. The Hill coefficient
for ATP binding to this mutant holoenzyme is 1.0 in contrast to 1.6 for the
native holoenzyme. The Kd (cAMP) is increased by approximately 1 order of
magnitude for both mutant forms of the holoenzyme in the presence of MgATP.
A similar shift is seen for the native holoenzyme. Further characterization
of the MgATP-binding properties of the wild-type holoenzyme indicates that
a binary complex containing catalytic subunit and MgATP is required, in
particular, for reassociation with the cAMP- bound regulatory subunit. This
binary complex is required for rapid dissociation of the bound cAMP and is
probably responsible for the observed reduction in cAMP-binding affinity
for the type I holoenzyme in the presence of MgATP.
Dissecting the domain structure of the regulatory subunit of cAMP- dependent protein kinase I and elucidating the role of MgATP
Department of Chemistry, University of California, La Jolla 92093.
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