title: Catalytic site amino acids of PKGI-alpha influence allosteric cGMP binding.
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.
authors.affiliation: Biology Department, Wheaton College, Wheaton, IL 60187, USA. jennifer.busch@wheaton.edu
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Busch
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Bridges
authors.forename: Robyn
authors.initials: R
authors.lastname: Richie-Jannetta
authors.forename: Brian P
authors.initials: BP
authors.lastname: Hollett
authors.forename: Sharron H
authors.initials: SH
authors.lastname: Francis
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D006152:Cyclic GMP / Q000737:chemistry* / Q000378:metabolism*; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D006152:Cyclic GMP