共查询到20条相似文献,搜索用时 23 毫秒
1.
Juliana Cheleski Ana Paula Citadini Maria Cristina Nonato 《Analytical biochemistry》2010,399(1):13-22
Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) catalyzes the oxidation of l-dihydroorotate to orotate with concomitant reduction of fumarate to succinate in the de novo pyrimidine biosynthetic pathway. Based on the important need to characterize catalytic mechanism of TcDHODH, we have tailored a protocol to measure TcDHODH kinetic parameters based on isothermal titration calorimetry. Enzymatic assays lead to Michaelis-Menten curves that enable the Michaelis constant (KM) and maximum velocity (Vmax) for both of the TcDHODH substrates: dihydroorotate (KM = 8.6 ± 2.6 μM and Vmax = 4.1 ± 0.7 μM s-1) and fumarate (KM = 120 ± 9 μM and Vmax = 6.71 ± 0.15 μM s-1). TcDHODH activity was investigated using dimethyl sulfoxide (10%, v/v) and Triton X-100 (0.5%, v/v), which seem to facilitate the substrate binding process with a small decrease in KM. Arrhenius plot analysis allowed the determination of thermodynamic parameters of activation for substrates and gave some insights into the enzyme mechanism. Activation entropy was the main contributor to the Gibbs free energy in the formation of the transition state. A factor that might contribute to the unfavorable entropy is the hindered access of substrates to the TcDHODH active site where a loop at its entrance regulates the open-close channel for substrate access. 相似文献
2.
Emily M.S. Hopwood Duale AhmedSusan M. Aitken 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(2):465-472
Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4–1.2 pH units, likely due to repositioning of the cofactor and modification of the pKa of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3–3, 26–58 and 124–568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The Kml-Cth of E333 substitution variants is increased ~ 17-fold, while Kml-OAS is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (kcat/Kml-OSHS = 7 ± 2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (kcat/Kml-Cth = 2100 ± 100) and 260-fold higher than that of l-Hcys (kcat/Kml-Hcys = 0.027 ± 0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity. 相似文献
3.
Lucena MN Garçon DP Mantelatto FL Pinto MR McNamara JC Leone FA 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2012,161(4):380-391
We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na+, K+)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg−1 H2O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg−1 H2O). Hemolymph [Na+] (323.0 ± 2.5 mmol L−1) and [Mg2+] (34.6 ± 1.0 mmol L−1) are hypo-regulated while [Ca2+] (22.5 ± 0.7 mmol L−1) is hyper-regulated; [K+] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L−1) but hypo-regulated (6.2 ± 0.7 mmol L−1) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na+, K+)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg−1; K0.5 = 7.07 ± 0.01 μmol L−1) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg−1; K0.5 = 0.11 ± 0.3 mmol L−1), both obeying cooperative kinetics, were disclosed. Modulation of (Na+, K+)-ATPase activity by Mg2+, K+ and NH4+ also exhibits site-site interactions, but modulation by Na+ shows Michaelis-Menten kinetics. (Na+, K+)-ATPase activity is synergistically stimulated up to 45% by NH4+ plus K+. Enzyme catalytic efficiency for variable [K+] and fixed [NH4+] is 10-fold greater than for variable [NH4+] and fixed [K+]. Ouabain inhibited ≈80% of total ATPase activity (KI = 464.7 ± 23.2 μmol L−1), suggesting that ATPases other than (Na+, K+)-ATPase are present. While (Na+, K+)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min−1 mg−1) and 45‰-acclimated crabs (around 154 nmol Pi min−1 mg−1), ATP affinity decreases 110-fold and Na+ and K+ affinities increase 2-3-fold in 45‰-acclimated crabs. 相似文献
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5.
Li QA Mavrodi DV Thomashow LS Roessle M Blankenfeldt W 《The Journal of biological chemistry》2011,286(20):18213-18221
PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismate-converting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 Å resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH3 to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rearrangements that establish an ammonia transport channel approximately 25 Å in length within each of the two MST/GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 Å resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn2+, Mn2+, and Ni2+ reduce the activity of PhzE. 相似文献
6.
J L Paukert J Henkin J Keesey J A DeMoss 《Archives of biochemistry and biophysics》1982,218(2):443-449
The multifunctional enzyme complex, anthranilate synthase from Neurospora crassa, irreversibly loses its glutamine-dependent anthranilate synthase activity on exposure to the reactive glutamine analogs DON and azaserine. Inactivation depends on the presence of the substrate chorismate, is enhanced by the cofactor Mg+2, and is antagonized by glutamine. Inactivation correlates well with the incorporation of [14C]DON into the protein with modification localized to the β subunit (Mr 84,000) of the complex, demonstrating directly that the β subunit provides the glutamine binding site for the glutamine-dependent anthranilate synthase reaction. The slower and less extensive loss of ammonia-dependent anthranilate synthase activity indicates that maximum expression of the ammonia-dependent anthranilate synthase activity by the α subunit also depends on the interaction with an active glutamine amidotransferase domain of the β subunit. 相似文献
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8.
Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg2+ and Na+ concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na+] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 ± 2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg2+] levels, which agrees quantitatively with the 32 ± 1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the KD value and Hill coefficient for [Mg2+]-dependent docking decrease significantly for [Na+] = 25 mM vs. 125 mM, indicating Mg2+ and Na+ synergy in the RNA folding process. 相似文献
9.
Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP+ or NAD+, but not NADPH and NADH, as pyridine nucleotide cofactor with Km values of 12 ± 2 and 390 ± 2 μM, compared to the Km for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The Km for corticosterone in the NADP+-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The Kcat values for nuclear activity with NADP+ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2. 相似文献
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11.
Otacilio C. Moreira 《BBA》2005,1708(3):411-419
The bidentate complex of ATP with Cr3+, CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca2+-ATPase and the Na+,K+-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca2+ and Na+, respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca2+-ATPase. The complex inhibited with similar efficiency the Ca2+-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T1/2 = 30 min at 37 °C) with a Ki = 28 ± 9 μM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg2+ but unaltered when Ca2+ was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca2+ occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La3+ with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca2+ at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca2+ promoted by the plasma membrane Ca2+-ATPase goes through an enzymatic phospho-intermediate that maintains Ca2+ ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP. 相似文献
12.
Pratik H. Lodha Emily M.S. HopwoodAdrienne L. Manders Susan M. Aitken 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(7):1424-1431
Cystathionine β-synthase (CBS) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue N84 of yeast CBS (yCBS), predicted to form a hydrogen bond with the hydroxyl moiety of the PLP cofactor, was mutated to alanine, aspartate and histidine. The truncated form of yCBS (ytCBS, residues 1-353) was employed in this study to eliminate any effects of the C-terminal, regulatory domain. The kcat/Kml-Ser of the N84A, N84D and N84H mutants for the β-replacement reaction is reduced by a factor of 230, 11000 and 640, respectively. Fluorescence resonance energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and N84A mutant, is altered in N84H and absent in N84D. PLP saturation values of 73%, 30% and 67% were observed for the alanine, aspartate and histidine mutants, respectively, compared to 98% for the wild-type enzyme. A marginal β-elimination activity was detected for N84D (kcat/Kml-Ser = 0.23 ± 0.02 M-1 s-1) and N84H (kcat/Kml-Ser = 0.34 ± 0.06 M-1 s-1), in contrast with wild-type ytCBS and the N84A mutant, which do not catalyze this reaction. The ytCBS-N84D enzyme is also inactivated upon incubation with l-serine, via an aminoacrylate-mediated mechanism. These results demonstrate that residue N84 is essential in maintaining the orientation of the pyridine ring of the PLP cofactor and the equilibrium between the open and closed conformations of the active site. 相似文献
13.
Franziska ExnowitzBernd Meyer Thomas Hackl 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(3):443-449
1H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, Km and Vmax, were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8 mM. Using the Lambert W function the parameters Km and Vmax were fitted to obtain the experimental progress curve and resulted in Km = 28 mM and Vmax = 13 μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding Km = 379 μM and kcat = 0.04 s− 1. The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function. 相似文献
14.
Nilusha Ragunathan Benjamin Pluvinage Elodie Sanfins Fernando Rodrigues-Lima Julien Dairou 《FEBS letters》2010,584(15):3366-29
Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg2+) and organic (CH3Hg+) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC50) = 250 nM and kinact = 1.4 × 104 M−1 s−1 for Hg2+ and IC50 = 1.4 μM and kinact = 2 × 102 M−1 s−1 for CH3Hg+). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC50 = 3 and 20 μM for Hg2+ and CH3Hg+, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1. 相似文献
15.
Dahmane Ouazia 《Analytical biochemistry》2010,398(1):45-563
α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (Km = 48 ± 5 μM, kcat = 291 ± 30 s−1), but turned over (2S)-ibuprofenoyl-CoA (Km = 86 ± 6 μM, kcat = 450 ± 14 s−1) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His6-tag had a catalytic efficiency (kcat/Km) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity. 相似文献
16.
Irina V. Nesmelova Elena Ermakova Mabel Pang Laura Lagartera Dolores Solís Linda G. Baum 《Journal of molecular biology》2010,397(5):1209-5276
Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K1 = 21 ± 6 × 103 M− 1) than the second (K2 = 4 ± 2 × 103 M− 1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K1 = 20 ± 10 × 103 M− 1 and K2 = 1.67 ± 0.07 × 103 M− 1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general. 相似文献
17.
Christine PeineltHans-Jürgen Apell 《Biophysical journal》2002,82(1):170-181
Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca2+ and H+ on binding of the other ion in the E1 and P-E2 states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg2+ a strictly competitive binding sequence, H2E1?HE1?E1?CaE1?Ca2E1, was found with two Ca2+ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K1/2(2 Ca) = 34 nM, K1/2(H) = 1 nM and K1/2(H2) = 1.32 μM. Up to 2 Mg2+ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K1/2(Mg) = 165 μM, K1/2(Mg2) = 7.4 mM). In the P-E2 state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca2+ and H+ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E2CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca2+ (0.4 mM and 25 mM) and H+ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E1 and P-E2 conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position. 相似文献
18.
Maximilian N. Kopylovich Kamran T. Mahmudov Konstantin V. Luzyanin 《Inorganica chimica acta》2011,374(1):175-579
(E)-2-(2-(2-hydroxyphenyl)hydrazono)-1-phenylbutane-1,3-dione (H2L) was synthesized by azocoupling of diazonium salt of 2-hydroxyaniline with 1-phenylbutane-1,3-dione and characterized by IR, 1H and 13C NMR spectroscopies and X-ray diffraction analysis. In solution, H2L exists as a mixture of the enol-azo and hydrazone tautomeric forms and a decrease of temperature and of solvent polarity shifts the tautomeric balance to the hydrazone form. In the solid state, H2L crystallizes from ethanol-water in the monohydrate hydrazone form, as shown by X-ray analysis. The dissociation constants of H2L (pK1 = 5.98 ± 0.04, pK2 = 9.72 ± 0.03) and the stability constants of its copper(II) complex (log β1 = 11.01 ± 0.07, log β2 = 20.19 ± 0.08) were determined by the potentiometric method in aqueous-ethanol solution. The copper(II) complex [Cu2(μ-L)2]n was isolated in the solid state and found by X-rays to be a coordination polymer of a binuclear core with a distorted square pyramidal metal coordination geometry. 相似文献
19.
Krzysztof L. Hyrc Akwasi Minta P. Rogelio Escamilla Patrick P.L. Chan Xenia A. Meshik Mark P. Goldberg 《Cell calcium》2013
Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca2+-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd = 0.49 ± 0.07 μM; ACR-1) or low affinity (Kd = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn2+ (Kd = 0.38 ± 0.02 nM) or Mg2+ (Kd ∼ 5 mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators. 相似文献