Adenosine Cyclic 3′,5′-Monophosphate Dependent Protein Kinase: Kinetic Mechanism for the Bovine Skeletal Muscle Catalytic Subunit

Paul F. Cook, Maynard E. Neville, Kent E. Vrana, F. Thomas Hartl, Robert Roskoski, Paul F. Cook

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Abstract

The kinetic mechanism for adenosine cyclic 3′,5′-monophosphate dependent protein kinase was determined from initial velocity studies in the absence and presence of the product MgADP and dead-end inhibitors. Data are consistent with random addition of MgATP and Ser-peptide and ordered release of phospho-Ser-peptide and MgADP with a dead-end E-MgADP-Ser-peptide complex. In addition to the metal required for the nucleotide, we also characterized the binding of Mg2+ to a second site. Increasing the Mg2+ results in a 5-6-fold decrease in Vmax in the presence or absence of 0.1 M KCl. There is a 5-fold increase in V/KMgATP in the absence of KCl and a 13-fold increase in V/KMgATP at 0.1 M KCl. The effect of increasing free Mg2+ on Vmax and V/K was also obtained with MgITP (20% the Vmax with MgATP) and MgGTP (10% the Vmax with MgATP) as substrates. The dissociation constant for Mg2+ from E-Ser-peptide-Mg2+ and central complexes is 2–3 mM. At low concentrations of free Mg2+ and no added KCl, competitive inhibitors of MgATP (Ki = 160 µM) decrease in the order adenosine = MgADP (Ki ≃ 40 µM) > AMP (Ki ≃ 8 mM). At saturating free Mg2+, Ki values are 10 µM (MgATP), 10 µM (MgADP), 40 µM (adenosine), and 850 µM (AMP). In addition, guanosine (1 mM) and MgPPPi (10 mM) failed to inhibit at low or high free Mg2+. A mechanism for nucleotide interaction is proposed on the basis of a hydrophobic site or negatively charged group in the region of the α-phosphate in the nucleotide binding site and the presence or absence of a negative charge on the α-phosphate. We also made use of a spectrophotometric assay for protein kinase that couples the production of MgADP to the pyruvate kinase and lactate dehydrogenase reactions. On the basis of determinations of the amount of phosphorylatable Ser-peptide by spectrophotometric or radioisotopic determinations, we find that commercial samples contain 25–50% nonpeptide material by weight.

Original languageEnglish (US)
Pages (from-to)5794-5799
Number of pages6
JournalBiochemistry
Volume21
Issue number23
DOIs
Publication statusPublished - Jan 1 1982

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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