Isotope partitioning in the adenosine 3',5'-monophosphate dependent protein kinase reaction indicates a steady-state random kinetic mechanism.
Isotope partitioning beginning with the binary E.MgATP and E.N-acetyl-Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ser-peptide) complexes indicates that the kinetic mechanism for the adenosine 3',5'-monophosphate dependent protein kinase is steady-state random. A total of 100% of the initial radioactive E.MgATP complex is trapped as phospho-Ser-peptide at infinite Ser-peptide concentration at both low and high concentration of uncomplexed Mg2+, suggesting that the off-rate of MgATP from the E.MgATP.Ser-peptide complex is slow relative to the catalytic steps. Km for Ser-peptide in the trapping reaction decreases from 17 microM at low Mg2+ to 2 microM at high Mg2+, indicating that Mg2+ decreases the off-rate for MgATP from the E.MgATP complex. A total of 100% of the radioactive E.Ser-peptide complex is trapped as phospho-Ser-peptide at low Mg2+, but only 40% is trapped at high Mg2+ in the presence of an infinite concentration of MgATP, suggesting that the off-rate for Ser-peptide from the central complex is much less than catalysis at low but not at high Mg2+. In support of this finding, the Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly (Ala-peptide) increases from 0.27 mM at low Mg2+ to 2.4 mM at high Mg2+. No trapping was observed at either high or low Mg2+ for the E.MgADP complex up to a phospho-Ser-peptide concentration of 5 mM. Thus, it is likely that in the slow-reaction direction the kinetic mechanism is rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D009206:Myocardium / Q000201:enzymology; D010761:Phosphorus Radioisotopes; D011494:Protein Kinases / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D014316:Tritium
D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D014316:Tritium; D000255:Adenosine Triphosphate; D011494:Protein Kinases