pH dependence of the 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 determine the acid-base chemical mechanism of the adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit, the pH dependence of the kinetic mechanism in the direction of MgADP phosphorylation has been determined using initial velocity studies in the presence and absence of dead-end inhibitors. The kinetic mechanism in the direction of MgADP phosphorylation is random, with changes in the preference of substrate binding as a function of pH. At pH 7.2 and below, the kinetic mechanism is ordered with phosphorylated peptide binding prior to MgADP. At pH 7.6, the opposite pathway with MgADP binding prior to phosphorylated peptide is preferred. The pH independence of V/Et is consistent with a mechanism in which reactants only bind to the correctly protonated form of the enzyme. The V/KMgADP value decreases, as the pH increases, giving a pK of about 7 which is likely that of a general acid, the same group that serves as a general base in the opposite reaction direction [Yoon, M.-Y., & Cook, P.F. (1987) Biochemistry 26, 4118]. The pKiPSP decreases at low pH giving a pK of about 6.5, probably reflecting the phosphate group of the peptide.
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Department of Biochemistry, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107.
Cook
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PF
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
D046911:Macromolecular Substances; D009842:Oligopeptides; D010748:Phosphopeptides; D000244:Adenosine Diphosphate; D011494:Protein Kinases