Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments.
Viscosogenic agents were used to test the diffusion limits of the reaction catalyzed by the catalytic subunit of the cAMP-dependent protein kinase. The effects of glycerol and sucrose on the maximum rate (kcat) and the apparent second-order rate constants (kcat/Kpeptide) for the phosphorylation of four peptidic substrates were measured at their pH optima. The agents were found to have moderate to no effect on kcat/Kpeptide for good and poor substrates, respectively. Conversely, kcat was highly sensitive to solvent viscosity for three of the four peptides at high concentrations of ATP. Taken together, these data indicate that enzymatic phosphorylation by the catalytic subunit proceeds with rapid or near rapid equilibrium binding of substrates and that all steps following the central substrate complex (i.e., chemical and conformational events) are fast relative to the rate-determining dissociation of product, ADP, when ATP levels are high. Under saturating concentrations of peptide I, LRRASLG, an unproductive form of the enzyme is populated. The observed phosphorylation rate from this complex is involved in rate limitation owing to a slow step separating unproductive and productive enzyme forms. The data are used to establish a kinetic mechanism for the catalytic subunit that predicts initial reaction velocities under varying concentrations of ATP and substrate.
Adams
J A
JA
Department of Chemistry, University of California, San Diego, La Jolla 92093-0654.
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014783:Viscosity
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases