Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase.
Site-directed mutagenesis was used to remove a critical phosphorylation site, Thr-197, near the active site of the catalytic subunit of cAMP-dependent protein kinase. This residue is present in a number of protein kinases, and its phosphorylation largely influences catalytic activity. We changed Thr-197 to aspartic acid and alanine and measured the effects of these substitutions on the kinetic mechanism and inhibitor affinities. The mutants were expressed as the free catalytic subunit and as soluble fusion proteins of glutathione-S-transferase. The values for KATP and Kpeptide for all three mutants are raised by approximately 2 orders of magnitude relative to the wild-type enzyme. Viscosometric measurements indicate that elevations in Kpeptide are the result of reduced rates for phosphoryl transfer and not reduced substrate affinities. This implies that the loop that contains the phosphothreonine, the activation loop, does not reduce access to the substrate site as proposed for the inactive forms of cdk2 kinase [DeBont, H. L., et al. (1993) Nature 363, 595-602] and MAP kinase [Zhang, F., et al. (1994) Nature 367, 704-711]. The mutants associate slowly with the wild-type regulatory subunit, although the cAMP-free wild-type regulatory subunit inhibits the mutants stoichiometrically. A mutant regulatory subunit that binds cAMP poorly and rapidly inhibits the wild-type catalytic subunit does not inhibit the mutant proteins. These data suggest that the phosphothreonine region serves as a docking surface for the regulatory subunit in the holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Adams
J A
JA
Department of Chemistry, San Diego State University, California 92182.
McGlone
M L
ML
Gibson
R
R
Taylor
S S
SS
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D013379:Substrate Specificity; D013816:Thermodynamics; D014783:Viscosity
D009842:Oligopeptides; D017868:Cyclic AMP-Dependent Protein Kinases