Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The modification and concomitant inactivation of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity analogs of peptide substrates potentially capable of undergoing disulfide interchange with enzyme-bound sulfhydryl groups have been used to probe the active site associated with peptide binding. The regeneration of catalytic activity on treatment of the modified enzymes with dithiothreitol and the observation that prior reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) blocks the modification of the kinase by these reagents are consistent with the proposal that only thiol residues are reacting. The affinity analog Leu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly, 1, and the closely related peptide AcLeu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly-OEt, 3, react with a single sulfhydryl as shown by the stoichiometry of the release of the 3-nitro-2-pyridinesulfenyl group and the amount of label incorporated in the enzyme when the radioactively labeled peptide analog of 3 (peptide 4) is employed as the modifying agent. The kinetics of the reaction of 1 with 4.3 microM catalytic subunit was monophasic (employing substrate in excess conditions), yielding an apparent value of KI of approximately 40 microM and a k2 value of approximately 0.25 s-1. The low value of the observed KI, together with the observation that protein kinase substrates inhibit the modification reactions, suggest strongly that the cysteine residue undergoing reaction is in the vicinity of the active site. By trypsin-catalyzed degradation and identification of the peptide segment modified by covalent attachment of the peptide portion of the radioactive analog 4, the single cysteine modified was identified as cysteine-198.
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PMID:Modification of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity labels related to peptide substrates. 628 62

The interaction of lin-benzoadenosine di- and triphosphates with the catalytic subunit and type II holoenzymes of adenosine cyclic 3',5'-monophosphate (cAMP) dependent protein kinase has been investigated by steady-state kinetics and fluorescence spectroscopy. lin-Benzo-ADP is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (8.0 microM) similar to the Ki for ADP (9.0 microM). This value agrees well with the Kd (9.0 microM) determined by fluorescence polarization titration. Type II holoenzymes from bovine brain and skeletal muscle have Kd values for lin-benzo-ADP of 3.4 microM and 3.5 microM, respectively, and each binds approximately 2 mol/mol of R2C2 tetramer. Furthermore, fluorescence polarization studies indicate that both the catalytic subunit and type II holoenzyme bind lin-benzo-ADP rigidly, so that there is little or no rotation of the lin-benzoadenine portion of the molecule within the nucleotide binding site. lin-Benzo-ATP is a substrate for the phosphotransferase activities of protein kinase with peptides, water, or type II regulatory subunit as phosphoryl acceptors. With Leu-Arg-Arg-Ala-Ser-Leu-Gly as phosphoryl acceptor, the Km for lin-benzo-ATP is 11.3 microM, and that for ATP is 11.9 microM. The Vmax with lin-benzo-ATP is 20% of the Vmax with ATP as the substrate [24.9 +/- 1.8 mumol/(min . mg) vs. 5.0 +/- 1.2 mumol/(min . mg)]. Thus lin-benzo-ATP is the best nucleotide substrate (besides ATP) for the catalytic subunit reported. 1,N6-Etheno-ATP (epsilon ATP), on the other hand, is a poor substrate for the catalytic subunit with a Km of 1.8 mM and a Vmax that is 4% of the Vmax for ATP, making it unsuitable as a fluorescence probe for cAMP-dependent protein kinase.
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PMID:Adenosine cyclic 3',5'-monophosphate dependent protein kinase: interaction of the catalytic subunit and holoenzyme with lin-benzoadenine nucleotides. 630 1

A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation.
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PMID:Characterization of a fluorescent substrate for the adenosine 3',5'-cyclic monophosphate-dependent protein kinase. 631 37

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanistic studies of cAMP-dependent protein kinase action. 636 50

cAMP-dependent protein kinase has been purified to homogeneity from adult bodies of Drosophila melanogaster. It is tetrameric in structure with two regulatory and two catalytic subunits that dissociate when activated by cAMP. The regulatory subunit exists in phospho and dephospho forms, which have electrophoretic mobilities in sodium dodecyl sulfate-polyacrylamide gels corresponding to Mr = 58,000 and 52,000, respectively. The catalytic subunit has a molecular weight of 40,000. The holoenzyme has a Stokes radius of 4.7 nm. The Km for activation by cAMP is substrate-dependent with Km values of 20 nM with histone H2B and 100 nM with the peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly. These physical and kinetic properties are very similar to those of the bovine heart Type II cAMP-dependent protein kinase. A Drosophila Type I cAMP-dependent protein kinase was also found in larval stages and during the first half of pupation but was absent in embryos and adults. The fly Type II enzyme was present in all developmental stages. Three regions of the Drosophila genome were found which, when present in three copies, significantly alter the specific activity of cAMP-dependent protein kinase. These are located at 29F-33F (30% increase), 46A-50C (17% increase), and 66B-67D (16% decrease).
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PMID:Drosophila cAMP-dependent protein kinase. 643 41

The kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase has been investigated employing the heptapeptide Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as substrate. Initial velocity measurements performed over a wide range of ATP and Kemptide concentrations indicated that the reaction follows a sequential mechanistic pathway. In line with this, the results of product and substrate inhibition studies, the patterns of dead end inhibition obtained employing the nonhydrolyzable ATP analogue, AMP X PNP (5'-adenylylimidodiphosphate), and equilibrium binding determinations, taken in conjunction with the patterns of inhibition observed with the inhibitor protein of the cAMP-dependent protein kinase that are reported in the accompanying paper (Whitehouse, S., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3682-3692), are best fit by a steady state Ordered Bi-Bi kinetic mechanism. Although the inhibition patterns obtained employing the synthetic peptide analogue in which the phosphorylatable serine was replaced by alanine were apparently incompatible with this mechanism, these inconsistencies appear to be due to some element of the structure of this latter peptide such that it is not an ideal dead end inhibitor substrate analogue. The data presented both here and in the accompanying paper suggest that both this substrate, analogue and the ATP analogue, AMP X PNP, do not fully mimic the binding of Kemptide and ATP, respectively, in their mechanism of interaction with the protein kinase. It is proposed that, as with some other kinase reactions, the configuration of the terminal anhydride bond of ATP assumes a conformation once the nucleotide is bound to the protein kinase that assists in the binding of either Kemptide or the inhibitor protein but not the alanine-substituted peptide and that AMP X PNP, because of its terminal phosphorylimido bond, cannot assume this conformation which favors protein (or peptide) binding.
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PMID:Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase. 683 26

Co3+ and Cr3+ complexes of beta, gamma-methylene-ATP (AMPPCP), which are substitution-inert substrate analogues inactive in phosphoryl transfer reactions, have been used in binding and structural studies of cAMP-dependent protein kinase. Dissociation constants of enzyme complexes with Co(NH3)4AMPPCP and CrAMPPCP and with Mn2+, which binds at an inhibitory site, were determined by electron paramagnetic resonance and by proton relaxation rate enhancement techniques. Nuclear relaxation rate measurements at 100 and 360 MHz were used to determine the distance between Mn2+ and the beta, gamma-methylene protons of Co-(NH3)4AMPPCP, yielding 7.4 +/- 0.6 A in the absence of enzyme and 5.0 +/- 0.9 A when both Mn2+ and Co-(NH3)4AMPPCP were bound to the enzyme. The effect of the paramagnetic CrAMPPCP on the electron spin relaxation time of the enzyme-bound Mn2+ was used used to calculate the distance between the two metal ions of 4.8 +/- 0.4 A. This distance and the Mn2+-methylene distance are consistent with the previous finding that the inhibitory metal bridges the enzyme to the triphosphate chain of the enzyme-bound nucleotide [Granot, J., Kondo, H., Armstrong, R. M., Mildvan, A. S., & Kaiser, E. T. (1979) Biochemistry 18, 2339]. From the paramagnetic effects on the relaxation rates of the protons of the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, distances from Mn2+ and Cr3+ to the serine methylene protons of 9.1 +/- 0.9 and 8.1 +/- 0.8 A, respectively, were calculated. These and previous measurements were used to estimate a distance of 5.3 +/- 0.7 A along the reaction coordinate between the gamma-phosphorus of ATP and the serine hydroxyl oxygen. This distance is 2 A greater than that required for molecular contact. The mechanistic implications of these findings are discussed.
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PMID:Magnetic resonance measurements of intersubstrate distances at the active site of protein kinase using substitution-inert cobalt(III) and chromium(III) complexes of adenosine 5'-(beta, gamma-methylenetriphosphate). 689 73

The regulatory subunit of cAMP-dependent protein kinase I has been cleaved proteolytically into two structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent, the two protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the two major domains, a 15-residue peptide that links the two domains has been isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1, susceptible to cleavage by both trypsin and thermolysin, has the following sequence: LysArg-Arg-Gly-Ala-Ile-Ser-Ala-. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site. The sequence at Site 2 was Val-Arg-Arg-Val-Ile-Ala. Cleavage here generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; however, in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. In contrast to the dissociated regulatory subunit, the holoenzyme is partially protected from proteolytic degradation.
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PMID:The structural domains of cAMP-dependent protein kinase I. Characterization of two sites of proteolytic cleavage and homologies to cAMP-dependent protein kinase II. 743 94

The effect of cyclic AMP (cAMP)-dependent phosphorylation and ADP-ribosylation on the activities of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), was investigated in order to determine the role of the N-terminus in covalent modification of the enzyme. The bifunctional enzyme was demonstrated to be a substrate in vitro for arginine-specific ADP-ribosyltransferase: 2 mol of ADP-ribose was incorporated per mol of subunit. The Km values for NAD+ and PFK-2/FBPase-2 were 14 microM and 0.4 microM respectively. A synthetic peptide (Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln) corresponding to the site phosphorylated by cAMP-dependent protein kinase was ADP-ribosylated on all three arginine residues. Analysis of ADP-ribosylation of analogue peptides containing only two arginine residues, with the third replaced by alanine, revealed that ADP-ribosylation occurred predominantly on the two most C-terminal arginine residues. Sequencing of the ADP-ribosylated native enzyme also demonstrated that the preferred sites were at Arg-29 and Arg-30, which are just N-terminal to Ser-32, whose phosphorylation is catalysed by cAMP-dependent protein kinase (PKA). ADP-ribosylation was independent of the phosphorylation state of the enzyme. Furthermore, ADP-ribosylation of the enzyme decreased its recognition by liver-specific anti-bifunctional-enzyme antibodies directed to its unique N-terminal region. ADP-ribosylation of PFK-2/FBPase-2 blocked its phosphorylation by PKA, and decreased its PFK-2 activity, but did not alter FBPase-2 activity. In contrast, cAMP-dependent phosphorylation inhibited the kinase and activated the bisphosphatase. These results demonstrate that ADP-ribosylation of arginine residues just N-terminal to the site phosphorylated by PKA modulate PFK-2 activity by an electrostatic and/or steric mechanism which does not involved uncoupling of N- and C-terminal interactions as seen with cAMP-dependent phosphorylation.
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PMID:Role of the N-terminal region in covalent modification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: comparison of phosphorylation and ADP-ribosylation. 761 45

Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src.
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PMID:The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src. 768 Nov 47


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