Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in Vmax without alterations in the Km for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.
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PMID:Phosphorylation and activation of tryptophan hydroxylase by exogenous protein kinase A. 859 57

Recombinant human liver phenylalanine hydroxylase (PAH) expressed in Escherichia coli has been purified to homogeneity. The recombinant enzyme exists in solution as a mixture of 80% tetramers and 20% dimers. A study of the kinetic properties of the enzyme indicates that compared to the recombinant and the native rat liver enzymes, the recombinant human enzyme is in an activated state. This conclusion is supported by the finding that its catalytic activity is only marginally stimulated by incubation with either phenylalanine or lysolecithin. In contrast, the native and the recombinant rat liver enzymes are activated 8- to 25-fold, respectively, when preincubated with phenylalanine or lysolecithin. In the absence of activators, the ratio of the hydroxylase activity in the presence of 6-methyl-5,6,7,8-tetrahydropterin compared to the activity in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), which is an index of the state of activation of the enzyme, is 4 for the human recombinant PAH compared to a value of 12 for the recombinant rat liver enzyme. Furthermore, the Km for phenylalanine in the presence of BH4 is 0.050 mM, a value that is one-fifth that of the recombinant rat liver enzyme. Covalent modification of the human enzyme by phosphorylation with protein kinase A provides further evidence that the human enzyme is in a substantially activated state. Phosphorylation, which results in the incorporation of 0.6 mol of phosphate/mol of subunit, leads to only a modest activation of 1.5-fold compared to about a 3-fold activation seen after phosphorylation of the native and the recombinant rat liver enzymes. Moreover, the recombinant human liver enzyme is less sensitive than the rat liver enzyme to stimulation by lysolecithin when tryptophan is the substrate. Just as is true for the rat liver enzyme, the apparent Km values for tryptophan and pheylalanine vary with the pterin cofactor employed. The ability of 7-tetrahydrobiopterin (7-BH4) to substitute for the natural cofactor tetrahydrobiopterin has been studied in vitro. The apparent Km for 7-BH4 for the recombinant human enzyme is 0.2 mM and the Km for phenylalanine is 0.05 mM. The hydroxylase reaction is severely inhibited by 7-BH4 in the presence of physiological concentrations of BH4. This inhibition can be overcome by a decrease in the concentration of phenylalanine. The implications of these novel properties of human PAH for phenylalanine homoestasis in man are discussed.
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PMID:Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties. 880 57

The antiviral activity of the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (PKR) is mediated through dsRNA binding leading to PKR autophosphorylation and subsequent inhibition of protein synthesis. Previous biochemical studies have suggested that autophosphorylation of PKR occurs via a protein-protein interaction and that PKR can form dimers in vitro. Using four independent biophysical and biochemical methods, we have characterized the solution complex formed between PKR and trans-activating region (TAR) RNA, a 57-nucleotide RNA species with double-stranded secondary structure derived from the human immunodeficiency virus type I genome. Chemical cross-linking and gel filtration analyses of PKR.TAR RNA complexes reveals that TAR RNA addition increases PKR dimerization and results in the formation of a solution complex with a molecular weight of approximately 150,000. Addition of TAR RNA to PKR results in a quenching of tryptophan fluorescence, indicative of a conformational shift. Through small angle neutron scattering analysis, we show that PKR exists in solution predominantly as a dimer, and has an elongated solution structure. Addition of TAR RNA to PKR causes a significant conformational shift in the protein at a 2:1 stoichiometric ratio of protein to RNA. Taken together, these data indicate that the PKR activation complex consists of a protein dimer bound cooperatively to one dsRNA molecule.
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PMID:Characterization of the solution complex between the interferon-induced, double-stranded RNA-activated protein kinase and HIV-I trans-activating region RNA. 908 92

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37 degrees C, TPH was much less stable (t1/2 = 28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by a t1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.
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PMID:A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity. 935 25

Calcium/calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to the enzyme and indirectly promotes the phosphorylation and synergistic activation of CaMKI by an exogenous kinase. We have evaluated the initial CaM-dependent activation of the unphosphorylated form of CaMKI. The kinetics of bacterially expressed human CaMKI show that the peptide syntide-2 is a relatively poor substrate, whereas the synapsin site-1 peptide is 17-fold more specific. The peptide ADR1G is 400-fold more specific than syntide-2, and its catalytic rate is among the highest reported for a kinase peptide substrate. To understand how CaM activates CaMKI, we have characterized the activation of the enzyme by CaM mutants with substitutions at hydrophobic residues. The point mutant M124Q located in the C-terminal domain of CaM produced a 57-fold increase in the CaM activation constant for CaMKI and suggests the involvement of methionine 124 in an important hydrophobic interaction with tryptophan 303 of CaMKI. Substituting two, three, and five hydrophobic residues in the N-terminal domain of CaM increased the CaM activation constant for CaMKI by 10-190-fold and lowered the maximal enzyme activity by more than 80%. Two of these N-terminal mutants of CaM do not affect the Km for peptide substrate but instead produce a 5-10-fold higher Km for ATP. This result demonstrates the critical role of the N-terminal domain of CaM in regulating the access of ATP to CaMKI.
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PMID:Characterization of substrate phosphorylation and use of calmodulin mutants to address implications from the enzyme crystal structure of calmodulin-dependent protein kinase I. 939 48

Many fungi undergo a morphological transition to filamentous growth in response to limiting nutrient conditions. Constitutively elongated Saccharomyces cerevisiae mutants (elm) have been isolated; the ELM1 gene encodes a putative serine/threonine protein kinase. A novel allele, elm1-15, has been isolated in an S288C-derived strain, which causes a pleiotropic phenotype, including media-specific growth effects, abnormal morphology and altered stress response, in cells that are auxotrophic for tryptophan. elm1-15 trp1 cells cannot use many nitrogen sources, are sensitive to amino acid analogues, have very low general amino acid permease activity and do not accumulate trehalose. In contrast, haploid elm1-15 TRP1 cells grow well in budding form on all media, are stress resistant and overaccumulate trehalose. Several lines of evidence suggest that Elm1 acts on functions related to the RAS/cAMP pathway. Overexpression of Elm1 partially rescues the ts phenotype of cdc25 and cyr1 mutants. Deletion of ELM1 in low PKA activity mutants increased the severity of their phenotypes, and activation of Ras2 decreases the cell elongation phenotype of elm1 mutants. A 'signal integration' model for the complex relationship of Elm1 and the RAS/cAMP pathway in controlling morphogenesis in response to nutrients is proposed.
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PMID:The control of morphogenesis in Saccharomyces cerevisiae by Elm1 kinase is responsive to RAS/cAMP pathway activity and tryptophan availability. 942 10

Oxygen free radicals may act as second messengers in signal transduction pathways and contribute to inflammatory diseases. We studied the action in vitro of radiolytically generated hydroxyl radicals (.OH) and superoxide radicals (O-2) on the cAMP-dependent protein kinases, I and II (PKAI and -II, respectively). The effects of the gasses O2 and N2O used to produce O-2 or .OH radicals by gamma-radiolysis of the water were also studied. PKAI is more sensitive than PKAII to oxygen gas (10 mM sodium formate) and to hydroxyl and superoxide radicals. Hydroxyl radicals decreased the kinase phosphotransferase activities stimulated either by cAMP or its site-specific analogs for both PKAI and PKAII; however, PKAI was more affected. The binding of [3H]cAMP and of 8-N3-[32P]cAMP to RI regulatory subunits was decreased. .OH caused a loss of tryptophan 260 fluorescence at site A of PKAI and of bityrosine production. Superoxide radicals affected only PKAI. O-2 modified both cAMP-binding sites A and B of the regulatory subunit but had a smaller effect on the catalytic subunit. The catalytic subunit was more sensitive to radicals when free than when part of the holoenzymes during exposure to the oxygen free radicals. These results suggest that oxygen free radicals alter the structure of PKA enzymes. Thus, oxidative modifications may alter key enzymes, including cAMP-dependent protein kinases, in certain pathological states.
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PMID:In vitro effects of oxygen-derived free radicals on type I and type II cAMP-dependent protein kinases. 971 18

The neurotransmitter serotonin has been implicated in numerous physiological functions and pathophysiological disorders. The hydroxylation of the aromatic amino acid tryptophan is rate-limiting in the synthesis of serotonin. Tryptophan hydroxylase (TPH), as the rate-limiting enzyme, determines the concentrations of serotonin in vivo. Relative serotonin concentrations are clearly important in neural transmission, but serotonin has also been reported to function as a local antioxidant. Identification of the mechanisms regulating TPH activity has been hindered by its low levels in tissues and the instability of the enzyme. Several TPH expression systems have been developed to circumvent these problems. In addition, eukaryotic expressions systems are currently being developed and represent a new avenue of research for identifying TPH regulatory mechanisms. Recombinant DNA technology has enabled the synthesis of TPH deletions, chimeras, and point mutations that have served as tools for identifying structural and functional domains within TPH. Notably, the experiments have proven long-held hypotheses that TPH is organized into N-terminal regulatory and C-terminal catalytic domains, that serine-58 is a site for PKA-mediated phosphorylation, and that a C-terminal leucine zipper is involved in formation of the tetrameric holoenzyme. Several new findings have also emerged regarding regulation of TPH activity by posttranslational phosphorylation, kinetic inhibition, and covalent modification. Inhibition of TPH by L-DOPA may have implications for depression in Parkinson's disease (PD) patients. In addition, TPH inactivation by nitric oxide may be involved in amphetamine-induced toxicity. These regulatory concepts, in conjunction with new systems for studying TPH activity, are the focus of this article.
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PMID:Advances in the molecular characterization of tryptophan hydroxylase. 977 Jun 40

The first step in the biosynthesis of melatonin in the pineal gland is the hydroxylation of tryptophan to 5-hydroxytryptophan. A cDNA of human tryptophan hydroxylase (TPH) was cloned from a library of human pineal gland and expressed in Escherichia coli. This cDNA sequence is identical to the cDNA sequence published from the human carcinoid tissue [1]. This human pineal hydroxylase gene encodes a protein of 444 amino acids and a molecular mass of 51 kDa estimated for the purified enzyme. Tryptophan hydroxylase from human brainstem exhibits high sequence homology (93% identity) with the human pineal hydroxylase. The recombinant tryptophan hydroxylase exists in solution as tetramers. The expressed human pineal tryptophan hydroxylase has a specific activity of 600 nmol/min/mg when measured in the presence of tetrahydrobiopterin and L-tryptophan. The enzyme catalyzes the hydroxylation of tryptophan and phenylalanine at comparable rates. Phosphorylation of the hydroxylase by protein kinase A or calmodulin-dependent kinase II results in the incorporation of 1 mol of phosphate/mol of subunit, but this degree of phosphorylation leads to only a modest (30%) increase in BH(4)-dependent activity when assayed in the presence of 14-3-3. Rapid scanning ultraviolet spectroscopy has revealed the formation of the transient intermediate compound, 4alpha-hydroxytetrahydrobiopterin, during the hydroxylation of either tryptophan or phenylalanine catalyzed by the recombinant pineal TPH.
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PMID:Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme. 1052 50

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. In whole cells and under certain circumstances in the cell-free system, the phosphorylation of p47phox mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47phox may be an important part of the activation mechanism. The total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47phox was substantially decreased, reflecting on the conformational change that occurs when p47phox was phosphorylated with protein kinase C. We show here that the phosphorylation of p47phox by protein kinase A or mitogen-activated protein kinase, however, had little effect on the intrinsic fluorescence of p47phox. In addition, the present experiments indicate that in the mutant p47phoxS379A, only the single S-->A mutation appears to be a major importance for the function of p47phox, which is able to undergo the change in conformation that takes place when p47phox is phosphorylated by protein kinase C.
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PMID:Kinase-dependent change in the conformation of the leukocyte NADPH oxidase subunit p47phox. 1067 33


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