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.cntdot.MgATP and E.cntdot.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.cntdot.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.cntdot.MgATP .cntdot.Ser-peptide complex is slow relative to the catalytic steps. Km for Ser-peptide in the trapping reaction decreases from 17 .mu.M at low Mg2+ to 2 .mu.M at high Mg2+, indicating that Mg2+ decreases the off-rate for MgATP from the E.cntdot.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 to 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.cntdot.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. Finally, no substrate inhibition was observed by Ser-peptide at any concentration of Mg2+ or MgATP when the coupled spectrophotometric assay with pyruvate kinase and lactate dehydrogenase was used.