REGULATION-DEFECTIVE MUTANTS OF TYPE-I CAMP-DEPENDENT PROTEIN-KINASE - CONSEQUENCES OF REPLACING ARGININE-94 AND ARGININE-95

Citation
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
Citations number
29
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
268
Issue
22
Year of publication
1993
Pages
16495 - 16503
Database
ISI
SICI code
0021-9258(1993)268:22<16495:RMOTCP>2.0.ZU;2-E
Abstract
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.