Cyclic AMP-dependent ATPase activity of bovine heart protein kinase.

Abstract

The adenosine 3',5'-monophosphate (cAMP) dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from bovine heart is characterized. That the ATPase activity is intimately associated with the catalytic subunit of the enzyme is suggested by the following: (i) the similar dependences of ATPase and protein kinase activities on cAMP; (ii) the dissociation of ATPase activity from the holoenzyme on addition of cAMP and its co-elution with the catalytic subunit on gel filtration chromatography; (iii) the similarity of the relative effectiveness of divalent metal ions in ATPase ;tnd protein kinase catalysis; and (iv) the correspondence of kinetically determined Km(MgATP) and Ki(MgDp) The original report (1) of an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity associated with cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from bovine brain and a similar finding with the catalytic subunit of the bovine liver enzyme (2) suggest that the catalysis of phosphoryl transfer to water might be a general phenomenon of diagnostic importance in the mechanistic study of protein phosphorylation by protein kinase. At variance with these observations are two other reports in which workers failed to detect ATPase activity in preparations of the bovine heart holoenzyme (3) and the catalytic subunit of the calf thymus enzyme (4). Before serious consideration of the mechanistic significance of the ATPase catalysis is undertaken, it is necessary to establish the intimacy of the association of ATPase and protein kinase activities. The present report describes the detection and characterization of an ATPase activity associated with the cAMP-dependent protein kinase from bovine heart. EXPERIMENTAL Protein kinase activity was assayed as described (5) or by monitoring phosphorylation of the synthetic peptidet substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (8). The ATPase activity was measured by the following modification of the previous thinlayer chromatography procedure (1). The origins on the polyethyleneimine thin-layer sheets were spotted with 5 Ml of 100 MM EDTA (pH 7.0) and dried with a stream of N2. Aliquots (5 Al) of reaction mixtures were spotted at each origin and dried. Five microliters of 2 mM unlabeled ATP/ADP was spotted at each origin and dried. This procedure allowed location of the nucleotides under UV light after chromatography. Spots were cut from the sheets and assayed for radioactivity in 6 ml of toluene/Omnifluor scintillant. ATPase assay mixtures contained 10 mM MgCl2 and 0.25 mg of bovine serum albumin per ml. All kinetic experiments were carried out at 250C in 50 mM Tris/150 mM KC1, pH 7.5, containing 0.2 mM dithiothreitol. Gel filtration chromatography was performed at 220C as described (5). Equilibrium dialysis was done at 22 +-1C for 24-36 hr. Partition of 3H-labeled nucleotides was monitored by liquid scintillation counting. RESULTSThe holoenzyme a...

Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

“…4B), as expected based on the crystal structure of PKR (Dar et al 2005). This K d is similar to that seen for ATP binding to other kinases such as PKA and MAPK (Armstrong et al 1979;Setyawan et al 1999), and in agreement with a value from Cole and coworkers of 20 6 2 mM, measured for ATP binding to K296R using competition fluorescence anisotropy experiments (Lemaire et al 2006). The second site was tighter in affinity (K d = 3.9 6 1.4 mM) but with a small binding stoichiometry of n = 0.11 6 0.09 (Fig.…”

Measurement of enzyme kinetics and inhibitor constants using enthalpy arrays

“…Reactions performed by combining a drop containing PKA and ATP with a drop containing Kemptide and ATP did not yield information on the K M of ATP (not shown). It is important to note that product inhibition by ADP ( K I = 7.3 μM; [23]) might a factor because the enzyme concentration used here in the initial drop (4 μM) was high enough that the intrinsic ATPase activity of PKA catalytic subunit (0.74 min −1 ; [25]) produced significant ADP in the time between mixing the enzyme with ATP and the addition of the Kemptide.…”

Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA

“…A similar approach was used to define the metal-binding sites in crystals of the porcine C subunit crystallized in the presence of inhibitor peptide and p[NH]ppA [7]. The metal requirements for the PKA C subunit were first described in detail by Armstrong et al [31], who showed that from two Mg 2+ -binding sites, one is required for catalysis and one is associated with inhibition. Based on crystal structures of the C subunit [7,8] and NMR studies [14] with inert complexes of ATP it was concluded that the activating metal bridges the βand γphosphates and involves an enzyme-ATP-metal bridge [14].…”