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1.
Methyltransferases possess a homologous domain that requires both a divalent metal cation and S-adenosyl-L-methionine (SAM) to catalyze its reactions. The kinetics of several methyltransferases has been well characterized; however, the details regarding their structural mechanisms have remained unclear to date. Using catechol O-methyltransferase (COMT) as a model, we perform discrete molecular dynamics and computational docking simulations to elucidate the initial stages of cofactor binding. We find that COMT binds SAM via an induced-fit mechanism, where SAM adopts a different docking pose in the absence of metal and substrate in comparison to the holoenzyme. Flexible modeling of the active site side-chains is essential for observing the lowest energy state in the apoenzyme; rigid docking tools are unable to recapitulate the pose unless the appropriate side-chain conformations are given a priori. From our docking results, we hypothesize that the metal reorients SAM in a conformation suitable for donating its methyl substituent to the recipient ligand. The proposed mechanism enables a general understanding of how divalent metal cations contribute to methyltransferase function. 相似文献
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C Tilgmann K Melen K Lundstr?m A Jalanko I Julkunen N Kalkkinen I Ulmanen 《European journal of biochemistry》1992,207(2):813-821
The rat and human recombinant soluble and membrane-bound catechol O-methyltransferase (S- and MB-COMT, respectively) were expressed using mammalian and baculovirus vectors. Low levels of rat and human S-COMT polypeptides were detected by immunoprecipitation in K-562 cell lines transfected with the S-COMT vectors. From K-562 cells transfected with the rat MB-COMT construct, both S- and MB-COMT recombinant proteins were detected by a rat COMT-specific anti-serum. Infection of lepidopteran Spodoptera frugiperda cells with recombinant S- or MB-COMT baculovirus constructs yielded high amounts of enzymically active and immunoreactive S- or MB-COMT proteins, respectively. Pulse/chase experiments with [35S]methionine-labelled insect cells infected with the MB-COMT baculovirus showed that the 30-kDa recombinant human MB-COMT polypeptide was not processed into the 25-kDa S-COMT form. Subcellular fractionations of insect cells, followed by immunoblotting with COMT antiserum, showed that recombinant S-COMT was found only in the soluble, cytoplasmic fraction, whereas MB-COMT resided both in soluble and membrane fractions. The recombinant MB-COMT sedimented in Percoll gradients at the density of 1.042 g/ml cosedimenting with the plasma-membrane marker. Fractionation and immunoblotting experiments on homogenized total rat brains indicated that the rat S-COMT (24 kDa) and some of the rat MB-COMT (28 kDa) was recovered in soluble fractions, whereas the microsomal material having COMT activity contained the MB-COMT polypeptide. The rat brain microsomal MB-COMT had a density of 1.042 g/ml in Percoll gradients, cosedimenting with the plasma-membrane and rough-endoplasmic-reticulum marker enzymes. The meta/para methylation ratio of dihydroxybenzoic-acid substrate by different recombinant and rat brain COMT-containing subcellular fractions was analysed. 相似文献
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Weinshilboum RM 《Cellular and molecular neurobiology》2006,26(4-6):539-561
1. Pharmacogenomics is the study of the role of inheritance in variation in the drug response phenotype-a phenotype that can vary from adverse drug reactions at one end of the spectrum to lack of therapeutic efficacy at the other. 2. The thiopurine S-methyltransferase (TPMT) genetic polymorphism represents one of the best characterized and most clinically relevant examples of pharmacogenomics. This polymorphism has also served as a valuable "model system" for studies of the ways in which variation in DNA sequence might influence function. 3. The discovery and characterization of the TPMT polymorphism grew directly out of pharmacogenomic studies of catechol O-methyltransferase (COMT), an enzyme discovered by Julius (Julie) Axelrod and his coworkers. 4. This review will outline the process by which common, functionally significant genetic polymorphisms for both COMT and TPMT were discovered and will use these two methyltransferase enzymes to illustrate general principles of pharmacogenomic research-both basic mechanistic and clinical translational research-principles that have been applied to a series of genes encoding methyltransferase enzymes. 相似文献
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Caged-Ca(2+) compounds such as nitrophenyl-EGTA (NP-EGTA) and DM-nitrophen (DMn) are extremely useful in biological research, but their use in live cells is hampered by cytoplasmic [Mg(2+)]. We determined the properties of Ca(2+) release from NP-EGTA and DMn by using Oregon green BAPTA-5N to measure changes in [Ca(2+)] after ultraviolet flash photolysis in vitro, with or without Mg(2+) present. A large fraction (65%) of NP-EGTA, which has a negligible Mg(2+) affinity, uncages with a time constant of 10.3 ms, resulting in relatively slow increases in [Ca(2+)]. Uncaging of DMn is considerably faster, but DMn has a significant affinity for Mg(2+) to complicate the uncaging process. With experimentally determined values for the Ca(2+) and Mg(2+) binding/unbinding rates of DMn and NP-EGTA, we built a mathematical model to assess the utility of NP-EGTA and DMn in rapid Ca(2+)-uncaging experiments in the presence of Mg(2+). We discuss the advantages and disadvantages of using each compound under different conditions. To determine the kinetics of Ca(2+) binding to biologically relevant Ca(2+) buffers, such as Ca(2+)-binding proteins, the use of DMn is preferable even in the presence of Mg(2+). 相似文献
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Using electric eel acetylcholinesterase (AChE) which was either membrane-bound (AChEm) or solubilized (AChEs), similar kinetics were seen in the absence of inhibitor or in the presence of edrophonium, trimethylammonium ion or paraoxon. Thus, both forms of the enzyme appear to behave similarly toward various inhibitors. However, in the presence of a probe sensitive to allosteric effects or changes in membrane fluidity, the two forms exhibit altered behavior. In the presence of F-, the relative rate of substrate hydrolysis by AChEm was reduced more rapidly than with AChEs, whether or not paraoxon was present. When inhibition by paraoxon (10(-7)-10(-4) M) was studied in the presence of F-, AChEs had a Hill coefficient of 1.0, whereas with AChEm the Hill coefficient changed from 0.8 to 1.5. 相似文献
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A soluble (100,000 x g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7. 5 and 35 degrees C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the K(m) for 6,7-dihydroxycoumarin was 500 +/- 21.5 microM, and that for S-adenosyl-L-methionine was 600 +/- 32.5 microM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-L-methionine, with a K(i) of 224 +/- 20.6 microM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg(2+), p-chloromercuribenzoic acid, and N-ethylmaleimide. 相似文献
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Kinetic studies of calcium and magnesium binding to troponin C 总被引:4,自引:0,他引:4
The kinetic mechanism of calcium binding was investigated for the high-affinity calcium-magnesium sites of troponin C (TN-C), for the C-terminal fragment containing only the high-affinity sites (TR2) and for the TN-C:TN-I (where TN-I represents the inhibitory subunit of troponin) complex. Rate constants were measured by the change in fluorescence of the proteins labeled with 4-(N-iodoacetoxyethyl-N-methyl-7-nitrobenz-2-oxa-1,3-diazole at Cys 98. Rate constants for calcium dissociation were also measured using the fluorescent calcium chelating agent quin 2. Calcium binding to TR2 at 4 degrees C is a two-step process at each binding site. (formula; see text) A first order transition (k1 = 700 s-1) follows the formation of a weakly bound collision complex (K0 = 2.5 X 10(3) M-1). The two sits of the labeled protein are distinguishable because of a 2-4-fold difference in rate constants of calcium dissociation. The kinetic evidence is consistent with additive changes in structure induced by calcium binding to two identical or nearly identical high-affinity sites. The mechanism for TN-C:TN-I is similar to TR2. TN-C gave complex kinetic behavior for calcium binding but calcium dissociation occurred with the same rate constants found for TR2. Calcium binding to the high-affinity sites of TnC can be interpreted by the same mechanism as for TR2 but an additional reaction possibly arriving from calcium binding to the low-affinity sites leads to a high-fluorescence intermediate state which is detected by the fluorophore. The interactions between the two classes of sites are interpreted by a model in which calcium binding at the high-affinity sites reverses the fluorescence change induced by calcium binding at the low-affinity sites. Magnesium binding to the calcium-magnesium sites of TR2 and TN-C occurs by the same two-step binding mechanism with a smaller value for K0 and a 5-fold larger rate constant of dissociation. 相似文献
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Analysis of competition binding between soluble and membrane-bound ligands for cell surface receptors
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Binding of the Fc portion of IgG coated on targets to Fcgamma receptors (e.g., CD16) expressed on leukocytes (i.e., 2D binding) is an initiating step for immune responses such as phagocytosis or antibody-dependent cellular cytotoxicity. In vivo, circulating leukocytes are exposed to plasma IgG. The competition from soluble IgG (i.e., 3D binding) may affect the 2D binding. Many cell surface receptors, CD16 included, have soluble counterparts. While their physiological significance is not clear, receptor-based competitive inhibition therapy, in which soluble receptors, ligands, and their analogs are employed to compete with surface-bound receptors and ligands to prevent unwanted adhesion, is widely used to treat various diseases. To provide a quantitative basis for design of these therapeutic approaches, we developed a mathematical model for 2D and 3D competition binding. The model relates cell-surface adhesion (in the presence and absence of dislodging forces) to the concentration of the soluble competitor, the densities of the surface-bound receptors and ligands, as well as the binding affinities of the 2D and 3D interactions. Binding of CD16-expressing cells to an IgG-coated surface in the presence of a soluble competitor (IgG or anti-CD16 antibody) was quantified by a centrifugation assay. The agreement between experiment and theory supports the validity of the model, which could be useful in predicting the efficacy of the competitor. 相似文献
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Pihlavisto P Reenilä I 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,781(1-2):359-372
Catechol O-methyltransferase (COMT) transfers a methyl group from S-adenosyl-L-methionine to the catechol substrate in the presence of magnesium. After the characterisation of COMT more than four decades ago, a wide variety of COMT enzyme assays have been introduced. COMT activity analysis usually consists of the handling of the sample and incubation followed by separation and detection of the reaction products. Several of these assays are validated, reliable and sensitive. Besides the studies of the basic properties of COMT, the activity assay has also been applied to explore the relation of COMT to various disease states or disorders. In addition, COMT activity analysis has been applied clinically since COMT inhibitors have been introduced as adjuvant drugs in the treatment of Parkinson's disease. 相似文献
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Ultraviolet resonance Raman study of drug binding in dihydrofolate reductase, gyrase, and catechol O-methyltransferase.
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This paper presents a study of the use of ultraviolet resonance Raman (UVRR) spectroscopic methods as a means of elucidating aspects of drug-protein interactions. Some of the RR vibrational bands of the aromatic amino acids tyrosine and tryptophan are sensitive to the microenvironment, and the use of UV excitation radiation allows selective enhancement of the spectral features of the aromatic amino acids, enabling observation specifically of their change in microenvironment upon drug binding. The three drug-protein systems investigated in this study are dihydrofolate reductase with its inhibitor trimethoprim, gyrase with novobiocin, and catechol O-methyltransferase with dinitrocatechol. It is demonstrated that UVRR spectroscopy has adequate sensitivity to be a useful means of detecting drug-protein interactions in those systems for which the electronic absorption of the aromatic amino acids changes because of hydrogen bonding and/or possible dipole-dipole and dipole-polarizability interactions with the ligand. 相似文献
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Kinetic mechanism of calcium binding to whiting parvalbumin 总被引:1,自引:0,他引:1
H D White 《Biochemistry》1988,27(9):3357-3365
Calcium binding to whiting parvalbumin induces large changes in the fluorescence, absorption, and circular dichroism spectra of the protein. The fluorescence emission maximum of the single tryptophan shifts from 325 to 348 nm upon the removal of calcium and decreases in intensity by 50%. All of the spectral changes are linear between 0 and 2 mol of calcium bound/mol of protein, which suggests that the only protein species present in significant concentration are PA0 and Pa-Ca2. The kinetics of calcium binding measured by stopped-flow fluorescence are accurately single exponential from 2 X 10(-7) to 2 X 10(-4) M free calcium. The kinetics of calcium dissociation show a pronounced lag and are best fit by two rate constants of 1.2 and 3.0 s-1. The minimal kinetic mechanism that adequately describes the rate and equilibrium data is a branched pathway mechanism in which the rate and equilibrium constants are markedly different for each pathway: (formula; see text) At [Ca] less than 2 microM the upper kinetic pathway of calcium binding predominates whereas at [Ca] greater than 2 microM calcium binding occurs predominantly by the lower kinetic pathway. Calcium dissociates primarily by the upper kinetic pathway. 相似文献
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S Schilling-Bartetzko A Bartetzko K H Nierhaus 《The Journal of biological chemistry》1992,267(7):4703-4712
Kinetic analyses of tRNA binding to the ribosome and of the translocation reaction showed the following results. 1) The activation energy for the P site binding of AcPhe-tRNA to poly(U)-programmed ribosomes is relatively high (Ea = 72 kJ mol-1; 15 mM Mg2+). If only the P site is occupied with deacylated tRNA(Phe), then the E site can be filled more easily with tRNA(Phe) (no activation energy measurable) than the A site with AcPhe-tRNA (Ea = 47 kJ mol-1; 15 mM Mg2+). 2) A ribosome with blocked P and E sites represents a standard state of the elongation cycle, in contrast to a ribosome with only a filled P site. The two states differ in that AcPhe-tRNA binding to the A site of a ribosome with prefilled P and E sites requires much higher activation energy (87 versus 47 kJ mol-1). The latter reaction simulates the allosteric transition from the post- to the pretranslocational state, whereby the tRNA(Phe) is released from the E site upon occupation of the A site (Rheinberger, H.-J., and Nierhaus, K. H. (1986) J. Biol. Chem. 261, 9133-9139). The reversed transition from the pre- to the posttranslocational state (translocation reaction) requires about the same activation energy (90 kJ mol-1). 3) Both elongation factors EF-Tu and EF-G drastically reduce the respective activation energies. 4) The rate of the A site occupation is slower than the rate of translocation in the presence of the respective elongation factors. The data suggest that the A site occupation rather than, as generally assumed, the translocation reaction is the rate-limiting step of the elongation cycle. 相似文献
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Aminu Usman Kevin Lobb Brett I. Pletschke Christopher G. Whiteley Brendan S. Wilhelmi 《Biochemistry and Biophysics Reports》2021
Catechol O-methyltransferase, an enzyme involved in the metabolism of catechol containing compounds, catalyzes the transfer of a methyl group between S-adenosylmethionine and the hydroxyl groups of the catechol. Furthermore it is considered a potential drug target for Parkinson’s disease as it metabolizes the drug levodopa. Consequently inhibitors of the enzyme would increase levels of levodopa. In this study, absorption, fluorescence and infrared spectroscopy as well as computational simulation studies investigated human soluble catechol O-methyltransferase interaction with silver nanoparticles. The nanoparticles form a corona with the enzyme and quenches the fluorescence of Trp143. This amino acid maintains the correct structural orientation for the catechol ring during catalysis through a static mechanism supported by a non-fluorescent fluorophore–nanoparticle complex. The enzyme has one binding site for AgNPs in a thermodynamically spontaneous binding driven by electrostatic interactions as confirmed by negative ΔG and ΔH and positive ΔS values. Fourier transform infrared spectroscopy within the amide I region of the enzyme indicated that the interaction causes relaxation of its β?structures, while simulation studies indicated the involvement of six polar amino acids. These findings suggest AgNPs influence the catalytic activity of catechol O-methyltransferase, and therefore have potential in controlling the activity of the enzyme. 相似文献
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Li Y Yang X Chang M Yager JD van Breemen RB Bolton JL 《Chemico-biological interactions》2005,152(2-3):151-163
Catechol O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. This enzyme is genetically polymorphic with a wild type and a variant form. Numerous epidemiological studies have shown that the variant form is associated with an increased risk of developing estrogen-associated cancers and a wide spectrum of mental disorders. There are seven cysteine residues in human S-COMT, all of which exist as free thiols and are susceptible to electrophilic attack and/or oxidative damage leading to enzyme inactivation. Here, the seven cysteine residues were systematically replaced by alanine residues by means of site-directed mutagenesis. The native forms and cysteine/alanine mutants were assayed for enzymatic activity, thermal stability, methylation regioselectivity, and reactivity of cysteine residues to thiol reagent. Our data showed that although there is only one encoding base difference between these two COMT forms, this difference might induce structural changes in the local area surrounding some cysteine residues, which might further contribute to the different roles they might play in enzymatic activity, and to the different susceptibility to enzyme inactivation. 相似文献
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Cobalamin binding activity occurred in the soluble fraction (69%) and the membrane fraction (31%) of Euglena mitochondria. The mitochondrial soluble cobalamin binding protein was purified about 580-fold in a yield of 34%; the membrane-bound cobalamin binding protein was solubilized with 2 M urea and partially purified. Both purified mitochondrial cobalamin binding proteins showed low pH dependency for activity. The pH optima of the soluble and membrane-bound cobalamin binding proteins were in the vicinity of 7.0 and 6.0–8.0, respectively. The K
s values of the soluble and membrane-bound cobalamin binding proteins for cyanocobalamin were 0.3 and 0.9 nM, respectively. Neither mitochondrial cobalamin binding proteins required metal ions for activity, but the activity of the soluble and membrane-bound cobalamin binding proteins was inhibited by 1 mM Mn2+, 48% and 89%, respectively. Molecular weight of the soluble cobalamin binding protein was calculated to be 93,000. The physiological roles of both mitochondrial cobalamin binding proteins were discussed on the basis of their properties and location in Euglena mitochondria.Abbreviations Cbl
cobalamin
- Ado-Cbl
5-deoxyadenosylcobalamin
- CN-Cbl
cyanocobalamin
- Me-Cbl
methylcobalamin
- OH-Cbl
hydroxocobalamin
- 2-AMP-Cbl
2-amino-2-methylpropanolylcobalamin 相似文献