Yj. Buechler et al., REGULATION-DEFECTIVE MUTANTS OF TYPE-I CAMP-DEPENDENT PROTEIN-KINASE - CONSEQUENCES OF REPLACING ARGININE-94 AND ARGININE-95, The Journal of biological chemistry, 268(22), 1993, pp. 16495-16503
The type Ialpha regulatory subunits of cAMP-dependent protein kinase c
ontain an autoinhibitor site, Arg-94-Arg-Gly-Ala-Ile, which serves as
a pseudosubstrate. To evaluate their contribution to subunit associati
on, Arg94 and Arg95, key determinants for peptide recognition, were re
placed singly and in tandem with Ala, Glu, and His. Unlike substrate p
eptides in which replacement of either arginine leads to an increase i
n K(m) of approximately 3 orders of magnitude, replacement of either a
rginine causes only a maximal 20-fold decrease in subunit association.
Replacement of both arginine residues with alanine, however, generate
s a regulatory subunit that can no longer recombine with the catalytic
subunit under physiological conditions when the regulatory subunit is
saturated with cAMP. To evaluate more fully the specific consequences
of replacing these 2 arginine residues, a rapid gel filtration chroma
tographic method was developed so that subunit affinity could be measu
red independently of assaying for catalytic activity. The R94,95A muta
nt shows a K(d(app)) = 677 nM, representing an increase of greater tha
n 3 orders of magnitude compared with the native subunits in the prese
nce of MgATP. In the absence of MgATP, the K(d(app)) for native regula
tory subunit was 125 nM, whereas the K(d(app)) for the R94,R95A mutant
regulatory subunit was determined to 2.87 muM. When this mutant holoe
nzyme is assayed at muM concentrations, no activity is observed, where
as below muM, activity is observed because of cAMP-independent subunit
dissociation.