Kinetic mechanism of the adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit in the direction of magnesium adenosine 5'-diphosphate phosphorylation.
In order to define the overall kinetic mechanism of adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit and also to elaborate the kinetic mechanism in the direction of peptide phosphorylation, we have determined its kinetic mechanism in the direction of MgADP phosphorylation. Studies of initial velocity as a function of uncomplexed Mg2+ (Mgf) in the absence and presence of dead-end inhibitors were used to define the kinetic mechanism. Data are consistent with the overall kinetic mechanism in the direction of MgADP phosphorylation being random with both the pathways allowed, i.e., the pathway in which MgADP binds to the enzyme prior to phosphorylated peptide and the pathway in which phosphorylated peptide binds to enzyme prior to MgADP. In addition, depending on the concentration of Mgf, one or the other pathway predominates. At low (0.5 mM) Mgf, the mechanism is steady-state ordered with the pathway in which phosphorylated peptide binds first being preferred; at high (10 mM) Mgf, the kinetic mechanism is equilibrium ordered, and the pathway in which MgADP binds first is preferred. This change in mechanism to equilibrium ordered at higher concentration of Mgf is due to an increase in affinity of the enzyme for MgADP and a decrease in affinity for the phosphorylated peptide. The Haldane relationship gives a Keq of 2 +/- 1 x 10(3) at pH 7.2, in agreement with the values obtained from 31P NMR (1.6 +/- 0.8 x 10(3)) and direct determination of reactant concentrations at equilibrium (3.5 +/- 0.6 x 10(3)).
Qamar
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Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107.
Yoon
M Y
MY
Cook
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PF
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D000242:Cyclic AMP / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*
D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D000242:Cyclic AMP; D011494:Protein Kinases