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1.
Ascenzi P Cao Y Tundo GR Coletta M Fanali G Fasano M 《Biochemical and biophysical research communications》2011,(1):185-189
Ferrous human serum heme–albumin (HSA–heme–Fe(II)) displays globin-like properties. Here, the effect of ibuprofen and warfarin on kinetics of HSA–heme–Fe(II) nitrosylation is reported. Values of the second-order rate constant for HSA–heme–Fe(II) nitrosylation (kon) decrease from 6.3 × 106 M−1 s−1 in the absence of drugs, to 4.1 × 105 M−1 s−1 and 4.8 × 105 M−1 s−1, in the presence of saturating amounts of ibuprofen and warfarin, respectively, at pH 7.0 and 20.0 °C. From the dependence of kon on the drug concentration, values of the dissociation equilibrium constant for ibuprofen and warfarin binding to HSA–heme–Fe(II) (i.e., K = 3.2 × 10−3 M and 2.6 × 10−4 M, respectively) were determined. The observed allosteric effects could indeed reflect ibuprofen and warfarin binding to the regulatory fatty acid binding site FA2, which brings about an alteration of heme coordination, slowing down HSA–heme–Fe(II) nitrosylation. Present data highlight the allosteric modulation of HSA–heme–Fe(II) reactivity by heterotropic effectors. 相似文献
2.
Two conserved non-canonical histidines are essential for activity of the cbb
3-type oxidase in Rhodobacter capsulatus 总被引:1,自引:0,他引:1
Cytochrome cbb
3 oxidase, a member of the heme–copper oxidase superfamily, catalyses the reduction of oxygen to water and generates a proton
gradient. Cytochrome c oxidases are characterized by a catalytic subunit (subunit I) containing two hemes and one copper ion ligated by six invariant
histidine residues, which are diagnostic of heme–copper oxidases in all type of the heme–copper oxidase superfamily. Alignments
of the amino acid sequences of subunit I (FixN or CcoN) of the cbb
3-type oxidases show that catalytic subunit also contains six non-canonical histidine residues that are conserved in all CcoN
subunits of the cbb
3 oxidase, but not the catalytic subunits of other members of heme–copper oxidases superfamily. The function of these six CcoN-specific
conserved histidines of cbb
3-type oxidase in R. capsulatus is unknown. To analyze the contribution of the two invariant histidines of CcoN, H300 and H394, in activity and assembly
of the Rhodobacter capsulatus
cbb
3-type oxidase, they were substituted for valine and alanine, respectively by site-directed mutagenesis. H300V and H394A mutations
were analyzed with respect to their activity and assembly. It was found that H394A mutation led to a defect in the assembly
of both CcoP and CcoO in the membrane, which results in almost complete loss of activity and that although the H300V mutant
is normally assembled in the membrane and retain their stability, its catalytic activity is significantly reduced when compared
with wild-type oxidase. 相似文献
3.
PriB is a primosomal protein required for the reinitiation of replication in bacteria. Here, we report the identification and characterization of a novel PriB protein in Klebsiella pneumoniae (KPN_04595; KpPriB). Unlike the well-studied Escherichia coli PriB protein (EcPriB), which exists as a homodimer comprising 104-aa polypeptides, KpPriB forms a monomer of only 55 aa, due to the absence of the 49 aa N-terminus in KpPriB. Although this N-terminal region (1–49 aa) in EcPriB contains several important residues, such as K18, R34, and W47, which are crucial for ssDNA binding, we found that KpPriB binds ssDNA, but not ssRNA, with comparable affinity as that for EcPriB. Results from filter-binding assays demonstrate that the KpPriB–ssDNA interaction is cooperative and salt-sensitive. Substituting the residue K33 in KpPriB with alanine, the position corresponding to the classic ssDNA-binding residue K82 of EcPriB located in loop L45, significantly reduced ssDNA-binding activity and cooperativity. These results reveal that the 1–49 aa region of the classical PriB protein is unnecessary for ssDNA binding. On the basis of these findings, the structure–function relationships of KpPriB are discussed. 相似文献
4.
Khajamohiddin Syed Chandramohan Kattamuri Thomas B. Thompson Jagjit S. Yadav 《Archives of biochemistry and biophysics》2011,(1):26
Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b5 reductase–cyt b5. In fungi, limited information is available for the cyt b5 reductase–cyt b5 system. Here we characterized the kinetic mechanism of (cyt b5r)–cyt b5 redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b5r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b5 reductases, Pc-cyt b5r lacked the typical ferricyanide reduction activity, a standard for cyt b5 reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b5r–cyt b5 complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b5r–cyt b5 complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b5r–cyt b5) in terms of supporting a P450 monooxygenase activity. 相似文献
5.
[Fe]-Hydrogenase catalyzes the reversible activation of H2. CO and CN− inhibit this enzyme with low affinity (Ki ≅ 0.1 mM) by binding to the iron site of the bound iron-guanyrylpyridinol cofactor. We report here that isocyanides, which are formally isoelectronic with CO and CN−, strongly inhibit [Fe]-hydrogenase (Ki as low as 1 nM). The [NiFe]- and [FeFe]-hydrogenases tested were not inhibited by isocyanides. UV–Vis and infrared spectra revealed that the isocyanides bind to the iron center of [Fe]-hydrogenase. The inhibition kinetics are in agreement with the proposed catalytic mechanism, including the open/closed conformational change of the enzyme. 相似文献
6.
Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed Km values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with Ki value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA. 相似文献
7.
Four brominated aliphatic hydrocarbons (1–4), including a novel brominated ene-tetrahydrofuran named as mutafuran H (1), and five sterols (5–9) were isolated from the South China Sea sponge Xestospongia testudinaria. The structure of 1 was determined on the basis of NMR (1H, 13C NMR, HSQC, HMBC, 1H–1H COSY, and NOESY), MS, and optical rotation analysis. Known compounds were identified by comparison of their NMR data with those reported in the literature. Compounds 1–4, and 6–9 were evaluated for their toxicity against Artemia salina larvae, and anti-acetylcholinesterase activity. 相似文献
8.
Hydroxylated metabolites of polychlorinated biphenyls (OHPCBs) interact with rat sulfotransferase 1A1 (rSULT1A1) as substrates and inhibitors. Previous studies have shown that there are complex and incompletely understood structure–activity relationships governing the interaction of rSULT1A1 with these molecules. Furthermore, modification of the enzyme with glutathione disulfide (GSSG) results in the conversion of some OHPCBs from inhibitors to substrates. We have now examined estimated values for the acid-dissociation constant (Ka) and the octanol–water distribution coefficient (D), as well as experimentally determined dissociation constants for enzyme complexes, to assist in the prediction of interactions of OHPCBs with rSULT1A1. Under reducing conditions, initial velocities for rSULT1A1-catalyzed sulfation exhibited a positive correlation with pKa and a negative correlation with log D of the OHPCBs. IC50 values of inhibitory OHPCBs decreased with decreasing pKa values for both the glutathione (GSH)-pretreated and GSSG-pretreated forms of rSULT1A1. Comparison of GSH- and GSSG-pretreated forms of rSULT1A1 with respect to binding of OHPCB in the presence and absence of adenosine 3′,5′-diphosphate (PAP) revealed that the dissociation constants with the two redox states of the enzyme were similar for each OHPCB. Thus, pKa and log D values are useful in predicting the binding of OHPCBs to the two redox forms of rSULT1A1 as well as the rates of sulfation of those OHPCBs that are substrates. However, the differences in substrate specificity for OHPCBs that are seen with changes in redox status of the enzyme are not directly related to specific structural effects of individual OHPCBs within inhibitory enzyme–PAP–OHPCB complexes. 相似文献
9.
Lombard M Salard I Sari MA Mansuy D Buisson D 《Archives of biochemistry and biophysics》2011,508(1):54-63
Fexofenadine, an antihistamine drug used in allergic rhinitis treatment, can be produced by oxidative biotransformation of terfenadine by Streptomyces platensis, which involves three consecutive oxidation reactions. We report here the purification and identification of the enzyme responsible for the first step, a cytochrome P450 (P450)-dependent monooxygenase. The corresponding P450, designated P450terf, was found to catalyze the hydroxylation of the t-butyl group of terfenadine and exhibited UV–Vis characteristics of a P450. Its interaction with terfenadine led to a shift of its Soret peak from 418 to 390 nm, as expected for the formation of a P450–substrate complex. In combination with spinach ferredoxin:NADP(+) oxidoreductase and ferredoxin, and in the presence of NADPH, it catalyzed the hydroxylation of terfenadine and some of its analogues, such as terfenadone and ebastine, with km values at the μM level, and kcat values around 30 min−1. Sequencing of the p450terf gene led to a 1206 bp sequence, encoding for a 402 aminoacid polypeptide exhibiting 56–65% identity with the P450s from the 107L family. These results confirmed that P450s from Streptomyces species are interesting tools for the biotechnological production of secondary metabolites, such as antibiotics or antitumor compounds, and in the oxidative biotransformation of xenobiotics, such as drugs. 相似文献
10.
Minoru Sakaguchi Takashi Matsuda Eiko Matsumura Tadashi Yoshimoto Masanori Takaoka 《Biochemical and biophysical research communications》2011,(4):693
Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24–72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10–60 μM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G0/G1 arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27kip1 and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G2/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G0/G1 arrest. These results indicate that POP is a part of the machinery that controls the cell cycle. 相似文献
11.
Dark-operative protochlorophyllide oxidoreductase, a nitrogenase-like enzyme, contains two [4Fe–4S] clusters, one in the L-protein ((BchL)2) and the other in the NB-protein ((BchN–BchB)2). The reduced NB-cluster in the NB-protein, which is ligated by 1Asp/3Cys residues, showed a broad S = 3/2 electron paramagnetic resonance signal that is rather rare in [4Fe–4S] clusters. A 4Cys-ligated NB-cluster in the mutated variant BchB–D36C protein, in which the Asp36 was replaced by a Cys, gave a rhombic normal S = 1/2 signal and lost the catalytic activity. The results suggest that Asp36 contributes to the low redox potential necessary to reduce protochlorophyllide. 相似文献
12.
Lemieux M Bouchard F Gosselin P Paquin J Mateescu MA 《Biochemical and biophysical research communications》2011,(3):429-434
The objective of this study was to evaluate the human NCI-N87 cell line as a model for gastric permeability drug studies under pH conditions of the stomach. The optimal conditions that led NCI-N87 cells to form a typical differentiated gastric epithelial barrier were a seeding density of 2.5 × 105 cells/cm2 on porous inserts and growth in serum-complemented RPMI-1640 medium until 18–27 days post-confluency. The resulting cell monolayers showed moderately high transepithelial electrical resistance (TEER) values of about 500 Ω cm2, cells of polygonal morphology expressing E-cadherin and ZO-1 proteins at their contact surfaces, and production of mucus clusters. The monolayers withstood apical pH of 7.4 down to 3.0 with the basal pH fixed at 7.4. The apparent permeability coefficients (Papp) of model compounds were evaluated in the apical-to-basolateral and basolateral-to-apical directions under different pH gradients. The monolayers were impermeable to the integrity marker Lucifer Yellow (low Papp of 0.3–1.1 × 10−6 cm/s). The furosemide Papp (0.4–1.5 × 10−5 cm/s) were slightly dependent on pH but remained moderate. The caffeine Papp (4.2–5.0 × 10−5 cm/s) were higher and insensitive to pH changes. The NCI-N87 cell line provides a useful in vitro tool to assess gastric drug permeability and absorption under physiologic conditions prevailing in the human stomach. 相似文献
13.
Ann Smith Jeffrey D. Eskew Corina M. Borza Michael Pendrak Richard C. Hunt 《Experimental cell research》1997,232(2):246
Heme–hemopexin supports and stimulates proliferation of human acute T-lymphoblastic (MOLT-3) cells, suggesting the participation of heme in cell growth and division. MOLT-3 cells express approximately 58,000 hemopexin receptors per cell (apparent Kd20 nM), of which about 20% are on the cell surface. Binding is dose- and temperature-dependent, and growth in serum-free IMDM medium is stimulated by 100–1000 nMheme–hemopexin, consistent with the high affinity of the receptor for hemopexin, and maximal growth is seen in response to 500 nMcomplex. Growth was similar in defined minimal medium supplemented with either low concentrations of heme–hemopexin or iron-transferrin, and either of these complexes were about 80% as effective as a serum supplement. Heme–hemopexin, but not apo–hemopexin, reversed the growth inhibition caused by desferrioxamine showing that heme–iron derived from heme catabolism is used for cell growth. Cobalt-protoporphyrin (CoPP)–hemopexin, which binds to the receptor but is not transported intracellularly [Smithet al.,(1993)J. Biol. Chem.268, 7365], also stimulated cell proliferation in serum-free IMDM but did not “rescue” the cells from desferrioxamine. Furthermore, CoPP–hemopexin effectively competed for the hemopexin receptor with heme–hemopexin and diminished its growth stimulatory effects. In addition, protein kinase C (PKC) is translocated to the plasma membrane within 5 min after heme–hemopexin is added to the medium, reaches maximum activity within 5–10 min, and declines to unstimulated levels by 30 min. Heme–hemopexin and CoPP-hemopexin both augmented MOLT-3 cell growth stimulated by serum. Thus, heme–hemopexin not only functions as an iron source for T-cells but occupancy of the hemopexin receptor itself triggers signaling pathway(s) involved in the regulation of cell growth. The stimulation of growth of human T-lymphocytes by heme–hemopexin is likely to be a physiologically relevant mechanism at sites of injury, infection, and inflammation. 相似文献
14.
Pan Y Abd-Rashid BA Ismail Z Ismail R Mak JW Pook PC Er HM Ong CE 《Chemico-biological interactions》2011,(1):1-8
Orthosiphon stamineus (OS) has been traditionally used to treat diabetes, kidney and urinary disorders, high blood pressure and bone or muscular pain. To assess the possibility of drug–herb interaction via interference of metabolism, effects of four OS extracts of different polarity and three active constituents (sinensetin, eupatorin and rosmarinic acid) on major human cDNA-expressed cytochrome P450 (CYP) enzymes were investigated. Three substrate-probe based high-performance liquid chromatography (HPLC) assays were established to serve as activity markers for CYP2C9, CYP2D6 and CYP3A4. Our results indicate that OS extracts and constituents exhibited differential modulatory effects on different CYPs. While none of the OS components showed significant inhibition on CYP2C9, eupatorin strongly and uncompetitively inhibited CYP2D6 activity with a Ki value of 10.2 μM. CYP3A4 appeared to be the most susceptible enzyme to OS inhibitory effects. It was moderately inhibited by OS dichloromethane and petroleum ether extract with mixed-type and noncompetitive inhibitions (Ki = 93.7 and 44.9 μg/mL), respectively. Correlation study indicated that the inhibition was accounted for by the presence of eupatorin in the extracts. When IC50 values of these extracts were expressed in volume per dose unit to reflect inhibitory effect at recommended human doses from commercially available products, moderate inhibition was also observed. In addition, CYP3A4 was strongly and noncompetitively inhibited by eupatorin alone, with a Ki value of 9.3 μM. These findings suggest that co-administration of OS products, especially those with high eupatorin content, with conventional drugs may have the potential to cause drug–herb interactions involving inhibition of major CYP enzymes. 相似文献
15.
Aerts D Verhaeghe TF Roman BI Stevens CV Desmet T Soetaert W 《Carbohydrate research》2011,346(13):1860-1867
In this study, the transglucosylation potential of six sucrose phosphorylase (SP) enzymes has been compared using eighty putative acceptors from different structural classes. To increase the solubility of hydrophobic acceptors, the addition of various co-solvents was first evaluated. All enzymes were found to retain at least 50% of their activity in 25% dimethylsulfoxide, with the enzymes from Bifidobacterium adolescentis and Streptococcus mutans being the most stable. Screening of the enzymes’ specificity then revealed that the vast majority of acceptors are transglucosylated very slowly by SP, at a rate that is comparable to the contaminating hydrolytic reaction. The enzyme from S. mutans displayed the narrowest acceptor specificity and the one from Leuconostoc mesenteroides NRRL B1355 the broadest. However, high activity could only be detected on l-sorbose and l-arabinose, besides the native acceptors d-fructose and phosphate. Improving the affinity for alternative acceptors by means of enzyme engineering will, therefore, be a major challenge for the commercial exploitation of the transglucosylation potential of sucrose phosphorylase. 相似文献
16.
Improved winter cold tolerance is widespread among small passerines resident in cold climates and is generally associated with elevated summit metabolic rate (Msum=maximum thermoregulatory metabolic rate) and improved shivering endurance with increased reliance on lipids as fuel. Elevated Msum and improved cold tolerance may result from greater metabolic intensity, due to mass-specific increase in oxidative enzyme capacity, or increase in the masses of thermogenic tissues. To examine the mechanisms underlying winter increases in Msum, we investigated seasonal changes in mass-specific and total activities of the key aerobic enzymes citrate synthase (CS) and β-hydroxyacyl CoA-dehydrogenase (HOAD) in pectoralis, supracoracoideus and mixed leg muscles of three resident passerine species, black-capped chickadee (Poecile atricapillus), house sparrow (Passer domesticus), and white-breasted nuthatch (Sitta carolinensis). Activities of CS were generally higher in winter than in summer muscles for chickadees and house sparrows, but not nuthatches. Mass-specific HOAD activity was significantly elevated in winter relative to summer in all muscles for chickadees, but did not vary significantly with season for sparrows or nuthatches, except for sparrow leg muscle. These results suggest that modulation of substrate flux and cellular aerobic capacity in muscle contribute to seasonal metabolic flexibility in some species and tissues, but such changes play varying roles among small passerines resident in cold climates. 相似文献
17.
The chlorophyll-deficient gun5-1 and cch Arabidopsis mutants carry single point mutations in the CHLH subunit of the magnesium chelatase enzyme, which catalyses the first committed step of chlorophyll biosynthesis. Recombinant Synechocystis ChlH subunits carrying the gun5-1 or cch mutations are inactive in Mg-chelatase assays, despite being able to bind both substrate and product, and retaining a capacity to form a ChlH–ChlI–ChlD Mg-chelatase complex. These mutant subunits act as inhibitors of ChlH, showing that the ChlH-porphyrin complex associates reversibly with the ChlI and D subunits during the catalytic cycle. This inhibition is reversed upon addition of Gun4. 相似文献
18.
Guesnet P Lallemand SM Alessandri JM Jouin M Cunnane SC 《Prostaglandins, leukotrienes, and essential fatty acids》2011,85(6):353-360
Background: We hypothesized that due to the absence of a dietary source of omega-3 fatty acids, the essential fatty acid (EFA) deficiency model leads to an overestimate of linoleic acid (LA) requirements. Methods: over 7 wk, young rats consumed an EFA diet containing either 0 en% linoleate (0LA) and 0 en% α-linolenate (0LNA) or a diet containing 0.5 en% LNA plus one of seven levels of added LA (0.12–4.0 en%; n=6/group).Results: Rats consuming the 0LA–0LNA diet had the lowest final body weight, 34–68% lower LA and arachidonate in plasma and liver, 87% lower LA in epididymal fat, and an 8–20 fold higher eicosatrienoate in plasma, liver and muscle lipids. 0.5LNA completely prevented the lower growth and partly prevented the rise in eicosatrienoate seen in the 0LA–0LNA group.Conclusion: Providing dietary LNA at 0.5 en% reduces the rat's physiological requirement for LA by an estimated factor of at least four (0.5 en% instead of 2 en%). Since LA requirements in humans are also based on the same flawed model of EFA deficiency, it is plausible that they too have been overestimated and should therefore be reinvestigated. 相似文献
19.
Lei J Mesters JR von Brunn A Hilgenfeld R 《Biochemical and biophysical research communications》2011,(4):680-685
This communication describes SAXS data based global structures of tetravalent antibody CD4–IgG2 and its dimeric to pentameric complexes with gp120s. Comparison of models brought forth that while the two CD4s grafted on each arm remain tightly packed in the unliganded antibody, they enable binding of first two gp120s preferentially to the same Fab arm in an asymmetric manner. Retention of residues in the CD4–Fab linker earlier reasoned to enable bi-fold collapse of gp120-bound soluble CD4, and observed asymmetry of the (CD4–IgG2)/(gp120)2 complex suggest that encoded flexibility in CD4–Fab linker is a critical structure–function factor for this broad spectrum neutralizing antibody. 相似文献
20.
Merly de Armas-Ricard Gloria Levicán Assaf Katz Jurgen Moser Dieter Jahn Omar Orellana 《Biochemical and biophysical research communications》2011,405(1):134
Glutamyl-tRNA reductase (GluTR) is the first enzyme committed to tetrapyrrole biosynthesis by the C5-pathway. This enzyme transforms glutamyl-tRNA into glutamate-1-semi-aldehyde, which is then transformed into 5-amino levulinic acid by the glutamate-1-semi-aldehyde 2,1-aminomutase. Binding of heme to GluTR seems to be relevant to regulate the enzyme function. Recombinant GluTR from Acidithiobacillus ferrooxidans an acidophilic bacterium that participates in bioleaching of minerals was expressed in Escherichia coli and purified as a soluble protein containing type b heme. Upon control of the cellular content of heme in E. coli, GluTR with different levels of bound heme was obtained. An inverse correlation between the activity of the enzyme and the level of bound heme to GluTR suggested a control of the enzyme activity by heme. Heme bound preferentially to dimeric GluTR. An intact dimerization domain was essential for the enzyme to be fully active. We propose that the cellular levels of heme might regulate the activity of GluTR and ultimately its own biosynthesis. 相似文献