Studies on the mechanism of phosphorylation of synthetic polypeptides by a calf thymus cyclic AMP-dependent protein kinase

Abstract

Synthetic polypeptides were employed as substrates in kinetic analyses of the reaction mechanism for the catalytic subunit of a cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from calf thymus. This enzyme preparation was shown to catalyze the transfer of phosphate from ATP to histone HI from calf thymus, as well as to two synthetic polypeptides, Arg-Lys-Ala-Ser-Gly-Pro (Hi-6) and Arg-Arg-Lys-Ala-Ser-Gly-Pro (Hi-7), corresponding to the amino acid sequence about serine-38 in calf HI. A related, basic heptapeptide corresponding to a sequence from pig liver pyruvate kinase, Leu-Arg-Arg-Ala-Ser-Leu-Gly (K), was also a substrate. The stoichiometry of peptide phosphorylation was established in each case as the transfer of 1 mol of phosphate from the y position of MgATP to the serine hydroxyl of 1 mol of the peptide. Steady-state, initial-velocity, kinetic parameters were determined for each substrate, using various concentrations of ATP. Under the conditions used, all synthetic peptides reacted with greater maximum velocities than whole histone HI. Nevertheless, the Km for HI, 54 ,uM, was lower than the Km values of the synthetic substrates. The most efficient substrate was peptide K, which had a V..l of 50.6,unmol/min per mg of kinase and a Km of 63 ;&M. In the absence of peptide substrate no ATPase activity was detectable at a sensitivity of 0.05% of the rate of peptide phosphorylation, suggesting that ATP is not cleaved to form an unstable phosphoenzyme-complex. The data are consistent with a sequential reaction mechanism involving a ternary complex between enzyme, polypeptide substrate, and ATP.Protein phosphorylation catalyzed by 3':5'-cyclic AMP (cAMP)-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) mediates many, if not all, of the effects of cAMP in eukaryotic cells (1). The use of synthetic polypeptides as substrates for cAMP-dependent protein kinase has thus far provided data concerning structure-efficacy relationships among phosphorylatable amino acid sequences (2-5). In particular, reports have concerned the minimum length of such sequences (2, 3) and the role of multiple basic residues on the NH2-terminal side of the phosphorylated serine in establishing substrate specificity (4, 5). Apart from modeling phosphorylation site specificity for protein kinases at the level of primary structure, these molecules are ideally suited for probing the catalytic mechanism of protein phosphorylation by a given kinase. The defined structure, homogeneity, and ease of manipulation of synthetic peptides allow a more accurate quantitation of catalytic parameters than can be obtained using intact protein substrates.In the present communication we examine the kinetic mechanism of peptide phosphorylation, using a highly purified protein kinase from calf thymus. We have synthesized hexaand heptapeptides corresponding to sequences about serine 38 in calf thymus histone HI, a reported site of cAMP-dependent

Cyclic AMP-dependent ATPase activity of bovine heart 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.…”

Localization of the High‐Affinity ATP Site in Adenosine‐3′:5′‐monophosphate‐Dependent Protein Kinase Type I

“…Furthermore no site for ATP in the catalytic subunit other than the active site has been found, as revealed by kinetic studies [23,24] and competition experiments with a variety of different analogues [9]. The idea of two different sites in the catalytic subunit for ATP, one site being completely abolished when the other is created, is fairly unlikely.…”

Autophosphorylation kinetics of protein kinases

“…In this model, the peptide binds to the catalytic site first, followed by ATP [6]. There have been numerous inconsistencies concerning the kinetic mechanism for the cAMP-dependent protein kinase-catalysed reaction [7][8][9][10][11][12][13]. Whitehouse et al [12] have carefully analysed the reaction mechanism using MgATP, peptide substrate, substrate analogues and protein kinase inhibitor.…”