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Catalytic site amino acids of PKGI-alpha influence allosteric cGMP binding
- Source :
- Frontiers in bioscience (Scholar edition). 5(2)
- Publication Year :
- 2013
-
Abstract
- Ser-64, an autophosphorylation site in the autoinhibitory subdomain of cGMP-dependent protein kinase type I-alpha (PKGI-alpha), lowers affinity for cGMP and suppresses catalytic activity (1). Using the structure of homologous cAMP-dependent protein kinase as a model, three conserved residues (Gln-401, His-404, Cys-518) in the PKGI-alpha catalytic site are predicted to be juxtaposed to Ser-64 (2). Individual point mutants (Q401A, H404A and C518A) and a double mutant (S64A/H404A) have been generated. cGMP or cAMP affinities (K(a)) of each mutant protein for phosphotransferase activation and allosteric (3H)cGMP-binding affinity (K(D)) of each mutant protein are significantly improved over those of wild-type (WT) PKGI-alpha. However, affinities (K(m)) of the mutant PKGs for peptide substrates or ATP are unaltered. Kinase activity ratio (-GMP/+cGMP) of H404A is greater than that for WT, Q401A, or C518A, and similar to that for S64A and S64A/H404A. These results reveal a unique mechanism whereby catalytic domain residues predicted to be spatially close to Ser-64 of the regulatory domain weaken the intrinsically high affinity of PKGI-alpha for cGMP and provide for autoinhibition of catalytic activity.
- Subjects :
- CGMP binding
General Immunology and Microbiology
biology
Chemistry
Stereochemistry
Autophosphorylation
Allosteric regulation
General Biochemistry, Genetics and Molecular Biology
Phosphotransferase
Kinetics
Structure-Activity Relationship
Allosteric enzyme
Mutant protein
Catalytic Domain
biology.protein
Mutagenesis, Site-Directed
Kinase activity
Amino Acids
Phosphorylation
Protein kinase A
Cyclic GMP
Cyclic GMP-Dependent Protein Kinase Type I
Signal Transduction
Subjects
Details
- ISSN :
- 19450524
- Volume :
- 5
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Frontiers in bioscience (Scholar edition)
- Accession number :
- edsair.doi.dedup.....bd2ffbe2f9ade35ec6a2e3524c2ff020