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1.
Hernández-Ortega A Lucas F Ferreira P Medina M Guallar V Martínez AT 《The Journal of biological chemistry》2011,286(47):41105-41114
Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O2 to H2O2 in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations show O2 access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O2 reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substitution of Phe-501 with tryptophan. The high oxygen catalytic efficiency of F501W, ∼2-fold that of native AAO and ∼120-fold that of F501A, seems related to a higher O2 availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O2 inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded >60% O2 population at 3–4 Å from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O2 reactivity of AAO decreased when the access channel was enlarged and increased when it was constricted by introducing a tryptophan residue. This is because the side chain of Phe-501, contiguous to the catalytic histidine (His-502 in AAO), helps to position O2 at an adequate distance from flavin C4a (and His-502 Nϵ). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O2 reactivity of this flavoenzyme. 相似文献
2.
Tongsook C Sucharitakul J Thotsaporn K Chaiyen P 《The Journal of biological chemistry》2011,286(52):44491-44502
p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxyphenylacetate to form 3,4-dihydroxyphenylacetate. Based on structures of the oxygenase component (C2), both His-120 and Ser-146 are located ∼2.8 Å from the hydroxyl group of HPA. The variants H120N, H120Q, H120Y, H120D, and H120E can form C4a-hydroperoxy-FMN (a reactive intermediate necessary for hydroxylation) but cannot hydroxylate HPA. The impairment of H120N is not due to substrate binding because the variant can still bind HPA. In contrast, the H120K variant catalyzes hydroxylation with efficiency comparable with that of the wild-type enzyme; the hydroxylation rate constant for H120K is 5.7 ± 0.6 s−1, and the product conversion ratio is 75%, compared with values of 16 s−1 and 90% for the wild-type enzyme. H120R can also catalyze hydroxylation, suggesting that a positive charge on residue 120 can substitute for the hydroxylation function of His-120. Because the hydroxylation reaction of wild-type C2 is pH-independent between pH 6 and 10, the protonation status of key components required for hydroxylation likely remains unchanged in this pH range. His-120 may be positively charged for selective binding to the phenolate form of HPA, i.e. to form the Hisδ+·HPAδ− complex, which in turn promotes oxygen atom transfer via an electrophilic aromatic substitution mechanism. Analysis of Ser-146 variants revealed that this residue is necessary for but not directly engaged in hydroxylation. Product formation in S146A is pH-independent and constant at ∼70% over a pH range of 6–10, whereas product formation for S146C decreased from ∼65% at pH 6.0 to 27% at pH 10.0. These data indicate that the ionization of Cys-146 in the S146C variant has an adverse effect on hydroxylation, possibly by perturbing formation of the Hisδ+·HPAδ− complex needed for hydroxylation. 相似文献
3.
Rinaldo-Matthis A Wetterholm A Martinez Molina D Holm J Niegowski D Ohlson E Nordlund P Morgenstern R Haeggström JZ 《The Journal of biological chemistry》2010,285(52):40771-40776
Human leukotriene C4 synthase (hLTC4S) is an integral membrane enzyme that conjugates leukotriene (LT) A4 with glutathione to form LTC4, a precursor to the cysteinyl leukotrienes (LTC4, LTD4, and LTE4) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC4S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3–99.9% of the specific activity against LTA4. Steady-state kinetics of R104A and R104S revealed that the Km for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λmax at 239 nm, could be detected using R104A or R104S hLTC4S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pKa to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA4. On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC4S by 88 and 70%, respectively, without significantly affecting the kcat/Km values for GSH, and a crystal structure of R31Q hLTC4S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC4S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases. 相似文献
4.
Michal R. Szymanski Maria J. Jezewska Wlodzimierz Bujalowski 《The Journal of biological chemistry》2010,285(13):9683-9696
Energetics and specificity of interactions between the Escherichia coli PriA helicase and the gapped DNAs have been studied, using the quantitative fluorescence titration and analytical ultracentrifugation methods. The gap complex has a surprisingly low minimum total site size, corresponding to ∼7 nucleotides of the single-stranded DNA (ssDNA), as compared with the site size of ∼20 nucleotides of the enzyme-ssDNA complex. The dramatic difference in stoichiometries indicates that the enzyme predominantly engages the strong DNA-binding subsite in interactions with the gap and assumes a very different orientation in the gap complex, as compared with the complex with the ssDNA. The helicase binds the ssDNA gaps with 4–5 nucleotides with the highest affinity, which is ∼3 and ∼2 orders of magnitude larger than the affinities for the ssDNA and double-stranded DNA, respectively. In the gap complex, the protein does not engage in cooperative interactions with the enzyme predominantly associated with the surrounding dsDNA. Binding of nucleoside triphosphate to the strong and weak nucleotide-binding sites of the helicase eliminates the selectivity of the enzyme for the size of the gap, whereas saturation of both sites with ADP leads to amplified affinity for the ssDNA gap containing 5 nucleotides and engagement of an additional protein area in interactions with the nucleic acid. 相似文献
5.
Goutam Chowdhury M. Wade Calcutt F. Peter Guengerich 《The Journal of biological chemistry》2010,285(11):8031-8044
Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH3CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect (Dkapp ∼ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The Dkapp for DEN was ∼3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO2H and CH3CO2H, respectively. In neither case was a lag observed, which was unexpected considering the kcat and Km parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde). 相似文献
6.
Jae Ho Seo Jung Chae Lim Duck-Yeon Lee Kyung Seok Kim Grzegorz Piszczek Hyung Wook Nam Yu Sam Kim Taeho Ahn Chul-Ho Yun Kanghwa Kim P. Boon Chock Ho Zoon Chae 《The Journal of biological chemistry》2009,284(20):13455-13465
Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine
thiol at their catalytic site. During peroxidase catalysis, the catalytic
cysteine, referred to as the peroxidatic cysteine (CP), cycles
between thiol (CP-SH) and disulfide (–S–S–)
states via a sulfenic (CP-SOH) intermediate. Hyperoxidation of the
CP thiol to its sulfinic (CP-SO2H) derivative
has been shown to be reversible, but its sulfonic
(CP-SO3H) derivative is irreversible. Our comparative
study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells
revealed that Prx II is more susceptible than Prx I to hyperoxidation and that
the majority of the hyperoxidized Prx II formation is reversible. However, the
hyperoxidized Prx I showed much less reversibility because of the formation of
its irreversible sulfonic derivative, as verified with
CP-SO3H-specific antiserum. In an attempt to identify
the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE
analysis, an N-acetylated Prx I was identified as part of the total
Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp
metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that
Nα-terminal acetylation (Nα-Ac) occurred
exclusively on Prx II after demethionylation. Nα-Ac of Prx II
blocks Prx II from irreversible hyperoxidation without altering its affinity
for hydrogen peroxide. A comparative study of
non-Nα-acetylated and Nα-terminal
acetylated Prx II revealed that Nα-Ac of Prx II
induces a significant shift in the circular dichroism spectrum and elevation
of Tm from 59.6 to 70.9 °C. These findings suggest
that the structural maintenance of Prx II by Nα-Ac may be
responsible for preventing its hyperoxidation to form
CP-SO3H.Peroxiredoxins
(Prxs)4 are a family
of peroxidases that possess a conserved cysteine residue at the catalytic site
for the reduction of peroxide/peroxynitrite. Using thiol-based reducing
equivalents, like thioredoxin, Prxs catalyze the reduction of hydrogen
peroxide, alkylhydroperoxides, and peroxynitrite to water, corresponding
alcohols, and nitrite, respectively
(1–8).
Based on the number and location of conserved cysteine residue(s) directly
involved in peroxide reduction, the six isotypes of mammalian Prx can be
grouped into three distinct subgroups as follows: 2-Cys Prx, atypical 2-Cys
Prx, and 1-Cys Prx,
(1–2,
5). Human Prx I (hPrx I) and
Prx II (hPrx II) are members of the 2-Cys Prx subgroup and thus contain two
conserved cysteine residues that are directly involved in peroxidase activity.
Cys52 for hPrx I and Cys51 for hPrx II are designated
the peroxidatic cysteines (CP). These residues attack the O–O
bond of the peroxide (ROOH) substrate to form the product (ROH) and the
sulfenic derivative CP-SOH. This sulfenic derivative then forms a
disulfide bond with the other conserved cysteine residue, which is referred to
as the resolving cysteine (CR; Cys173 in hPrx I and
Cys172 in hPrx II). In the case of 2-Cys Prxs, the disulfide
partners, CP and CR, reside within different subunits;
therefore, the disulfide bond established between CP and
CR (CP-S–S-CR) is intermolecular. The
reduced thioredoxin molecule is responsible for reducing the
CP-S–S-CR disulfide bond to generate sulfhydryls
(1–3,
5,
9).The CP of eukaryotic 2-Cys Prxs is vulnerable to hyperoxidation,
which results in the loss of its peroxidase activity. This feature is referred
to as the “floodgate” mechanism, by which Prxs function as a redox
sensor for the regulation of cell signaling
(10–11).
Hyperoxidation of CP does not occur when the disulfide bond
(CP-S–S-CR) is formed. However, the thiol
(CP-SH) can be hyperoxidized via the sulfenic (CP-SOH)
derivative intermediate in the absence of CP-S-S-CR
formation during catalysis
(12). Two different
hyperoxidation products of CP, the reversible sulfinic
(CP-SO2H) derivative and the irreversible sulfonic
(CP-SO3H) derivative, have been identified. The
irreversible CP-SO3H was reported in Tsa1p, a yeast
2-Cys Prx, based on in vivo and in vitro regeneration assay
results, and a stronger reactivity to an anti-Tsa1p-SO3H antibody,
which exhibits high specificity toward Tsa1p-CP-SO3H
relative to Tsa1p-CP-SO2H
(13). Both forms of
hyperoxidized Prxs, CP-SO2H and
CP-SO3H, are superimposed on the acidic migrated spot
instead of the Prx-SH spot on a two-dimensional polyacrylamide gel because of
the introduction of one negative charge by hyperoxidation
(12–16).
The protein sulfinic acid reductase, sulfiredoxin, is responsible for
reversing 2-Cys Prx-SO2H to Prx-SH in the presence of ATP and
thiol-reducing equivalents like thioredoxin or glutathione
(17–24).
Until now, an intracellular enzymatic regeneration system for
Prx-SO3H has not been reported.Because mammalian Prx I and Prx II have been studied independently in a
number of different organisms and cultured cells, the comparative biochemical
data supporting their distinctive functional identities is still very limited.
Recombinant Prx I (rPrx I) showed a 2.6-fold higher specific activity as a
peroxidase than the recombinant Prx II (rPrx II) without any obvious catalytic
or mechanistic differences
(25,
26). Recent competition
kinetics studies of hPrx II revealed a rate constant of 1.3 ×
107 m–1 s–1, which is
fast enough to favor an intracellular hydrogen peroxide target even in
competition with catalase or glutathione peroxidase
(27,
28). The kinetic parameters of
the competition assay for hPrx I are still not available. Mammalian Prx I
interacts with and regulates a broad spectrum of proteins, such as the Src
homology domain 3 of c-Abl
(29), the Myc
box II (MBII) domain of c-Myc
(30), the
macrophage migration inhibitory factor (MIF,
31), the androgen receptor
(32), and the
apoptosis signal-regulating
kinase-1 (ASK-1)
(33). The suggested roles of
Prx I in interactions with these molecules are those of a tumor repressor, a
survival enhancer, and a growth regulator. Although these suggested functions
are controversial (34), all of
them can be attributed to the peroxide-scavenging capacity of Prx I (at least
in part), except for the enhancement of androgen receptor transactivation
(32). Prx II interacts with
platelet-derived growth factor receptor and functions as a negative regulator
for platelet-derived growth factor signaling
(35). Prx II also binds to
phospholipase D1 (PLD1) and functions as a
hydrogen peroxide-stimulated PLD1 signal terminator
(36). Both of these suggested
Prx II roles are attributable to the peroxidase activity of Prx II. The major
phenotypes of Prx I knock-out mice involve the development of a variety of
age-related cancers, hemolytic anemia
(37), and dramatic shifts in
subcellular reactive oxygen species localization
(38). Prx II knock-out mice
exhibit splenomegaly and a lack of tumor development in any cell type or
tissue (39). Until now, the
protein molecule that interacts with Prx I and Prx II has not been
characterized, and there is no indication of a heterodimer between Prx I and
Prx II. Despite their similar peroxide-scavenging capacities, it is reasonable
to conclude that the Prx I and Prx II are unable to compensate for each other
in terms of physiological roles. There are several examples of tissue- or cell
type-specific expression patterns, such as exclusive Prx I expression in
astrocytes and Leydig cells and Prx II expression in neurons and Sertoli cells
(40,
41); however, Prx I and Prx II
are coexpressed in the majority of mammalian cells and tissues, suggesting
distinguished biochemical characteristics of their cellular regulatory
mechanisms. Recently, the unique presence of Cys83 in hPrx I, which
contributes to the stability of the dimer-dimer interface and suppresses local
unfolding, has been claimed to be prone to overoxidation of Prx I
(42). The contribution of the
dimer-decamer interconversion to the regulation of Prx I activity has also
been reported (43).In this study, we confirmed that hPrx II was more susceptible to
hyperoxidation as well as more prone to regeneration than hPrx I in HeLa
cells. We also found that the difficulty in regenerating hPrx I was caused by
irreversible sulfonic (CP-SO3H) hyperoxidation. Using
AspN (EC 3.4.24.33) peptide fingerprints, we identified the
Nα-terminal acetylation exclusively on hPrx II. In addition,
we provide evidence for the structural maintenance offered by
Nα-terminal acetylation of hPrx II, which possibly
contributes to preventing irreversible overoxidation of
CP-SO3H. 相似文献
7.
Thotsaporn K Chenprakhon P Sucharitakul J Mattevi A Chaiyen P 《The Journal of biological chemistry》2011,286(32):28170-28180
p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C2), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ∼30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H2O2 elimination. Studies on pH dependence showed that the rate constant of H2O2 elimination in H396N and H396V increased when pH increased with pKa >9.6 and >9.7, respectively, similar to the wild-type enzyme (pKa >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H2O2 elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H2O2 elimination in S171A and S171T were ∼1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H2O2 elimination increased with pH rise and exhibited an approximate pKa of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C2. 相似文献
8.
Melissa M. Stacey Alexander V. Peskin Margreet C. Vissers Christine C. Winterbourn 《Free radical biology & medicine》2009,47(10):1468-1476
Peroxiredoxin 2 (Prx2) is an abundant thiol protein that is readily oxidized in erythrocytes exposed to hydrogen peroxide. We investigated its reactivity in human erythrocytes with hypochlorous acid (HOCl) and chloramines, relevant oxidants in inflammation. Prx2 was oxidized to a disulfide-linked dimer by HOCl, glycine chloramine (GlyCl), and monochloramine (NH2Cl) in a dose-dependent manner. In the absence of added glucose, Prx2 and GSH showed similar sensitivities. Second-order rate constants for the reactions of Prx2 with NH2Cl and GlyCl were 1.5 × 104 and 8 M−1 s−1, respectively. The NH2Cl value is 10 times higher than that for GSH, whereas Prx2 is 30 times less sensitive than GSH to GlyCl. Thus, the relative sensitivity of Prx2 to GlyCl is greater in the erythrocyte. Oxidation of erythrocyte Prx2 and GSH was less in the presence of glucose, probably because of recycling. High doses of NH2Cl resulted in incomplete regeneration of reduced Prx2, suggesting impairment of the recycling mechanism. Our results show that, although HOCl and chloramines are less selective than H2O2, they nevertheless oxidize Prx2. Exposure to these inflammatory oxidants will result in Prx2 oxidation and could compromise the erythrocyte's ability to resist damaging oxidative insult. 相似文献
9.
Storbeck S Saha S Krausze J Klink BU Heinz DW Layer G 《The Journal of biological chemistry》2011,286(30):26754-26767
During the biosynthesis of heme d1, the essential cofactor of cytochrome cd1 nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate. 相似文献
10.
Faik N. Musayev Martino L. Di Salvo Mario A. Saavedra Roberto Contestabile Mohini S. Ghatge Alexina Haynes Verne Schirch Martin K. Safo 《The Journal of biological chemistry》2009,284(45):30949-30956
Mutations in pyridoxine 5′-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5′-phosphate oxidase catalyzes the oxidation of pyridoxine 5′-phosphate to pyridoxal 5′-phosphate, the active cofactor form of vitamin B6 required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is ∼850-fold less efficient than the wild-type enzyme due to an ∼192-fold decrease in pyridoxine 5′-phosphate affinity and an ∼4.5-fold decrease in catalytic activity. There is also an ∼50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 Å crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a β-strand-loop-β-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5′-phosphate oxidase mutations. 相似文献
11.
1. Thiol oxidation by a lipid peroxide or hydrogen peroxide was as efficient in denatured non-haem proteins as in small thiols. Both peroxides were relatively ineffective in oxidizing haemoprotein thiols, especially at low pH. Increased amounts of haematin decreased greatly the efficiency of GSH oxidation by peroxides especially at low pH. 2. Other than the haematin ring, the thiol group was found to be probably the group in proteins most sensitive to modification by peroxides. 3. At low concentrations, the fatty acid moiety of a lipid peroxide appeared to impede thiol oxidation in proteins, probably by hydrophobic bonding to the protein, rather than to stimulate thiol oxidation by denaturing the protein and thereby increasing the exposure and reactivity of the thiol group. 4. The relative rates of thiol oxidation by peroxides in the different thiols were: haemoprotein thiols>small thiols>other protein thiols. In all cases, thiol oxidation was much more rapid by the lipid peroxide than by hydrogen peroxide. 相似文献
12.
Likui Yang Chandrashekhara Manithody Shabir H. Qureshi Alireza R. Rezaie 《The Journal of biological chemistry》2010,285(37):28488-28495
The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with ∼40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31–41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited ∼5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with ∼4-fold decreased kcat and ∼2-fold decreased Km, although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X. 相似文献
13.
Chun-Ho Wong Yuan Fu Sreenivasa Rao Ramisetty Anne M. Baranger Steven C. Zimmerman 《Nucleic acids research》2011,39(20):8881-8890
Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disease caused by expanded CCUG repeats that may exhibit toxicity by sequestering the splicing regulator MBNL1. A series of triaminotriazine- and triaminopyrimidine-based small molecules (ligands 1–3) were designed, synthesized and tested as inhibitors of the MBNL1–CCUG interaction. Despite the structural similarities of the triaminotriazine and triaminopyrimidine units, the triaminopyrimidine-based ligands bind with low micromolar affinity to CCUG repeats (Kd ∼ 0.1–3.6 µM) whereas the triaminotriazine ligands do not bind CCUG repeats. Importantly, these simple and small triaminopyrimidine ligands exhibit both strong inhibition (Ki ∼ 2 µM) of the MBNL1–CCUG interaction and high selectivity for CCUG repeats over other RNA targets. These experiments suggest these compounds are potential lead agents for the treatment of DM2. 相似文献
14.
Gallardo-Soler A Gómez-Nieto C Campo ML Marathe C Tontonoz P Castrillo A Corraliza I 《Molecular endocrinology (Baltimore, Md.)》2008,22(6):1394-1402
Macrophages are phagocytic cells that play essential roles in innate immunity and lipid homeostasis. The uptake of modified lipoproteins is an important early event in the development of atherosclerosis. We analyzed the ability of modified low-density lipoprotein (LDL) (oxidized and acetylated) to alter the expression and activity of arginases (ArgI and ArgII) in macrophages. We show that ArgI expression is potently induced by both oxidized and acetylated LDL in macrophages. We further show that this effect is mediated by peroxisome proliferator-activated receptors (PPAR). ArgI expression is highly responsive to agonists for PPARgamma and PPARdelta but not PPARalpha. Moreover, the induction of ArgI by both PPAR agonists and IL-4 is blocked in macrophages from PPARgamma- and PPARdelta-deficient mice. Functionally, PPAR activity induces macrophage activation toward a more Th2 immune phenotype in a model of Leishmania major infection. We show that PPARgamma and -delta ligands promote intracellular amastigote growth in infected macrophages, and this effect is dependent on both PPAR expression and Arg activity. Collectively, our results strongly suggest that ArgI is a key marker of the alternative program triggered by PPAR in macrophages. 相似文献
15.
María Morales María J. Mate Antonio Romero María Jesús Martínez ángel T. Martínez Francisco J. Ruiz-Due?as 《The Journal of biological chemistry》2012,287(49):41053-41067
Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the
ability to oxidize Mn2+ and high redox potential aromatic
compounds, respectively. Moreover, it is also able to oxidize phenols (and low
redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A
high efficiency site (with 2,6-dimethoxyphenol and
p-hydroquinone catalytic efficiencies of ∼70 and
∼700 s−1 mm−1, respectively)
was localized at the same exposed Trp-164 responsible for high redox potential
substrate oxidation (as shown by activity loss in the W164S variant). The second
site, characterized by low catalytic efficiency (∼3 and ∼50
s−1 mm−1 for
2,6-dimethoxyphenol and p-hydroquinone, respectively) was
localized at the main heme access channel. Steady-state and transient-state
kinetics for oxidation of phenols and dyes at the latter site were improved when
side chains of residues forming the heme channel edge were removed in single and
multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and
E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel
similar to those of the exposed tryptophan site. The heme channel enlargement
shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants
would allow a better substrate accommodation near the heme, as revealed by the
up to 26-fold lower Km values (compared with native
VP). The resulting interactions were shown by the x-ray structure of the
E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43
and Pro-139 in the distal heme pocket (at the end of the heme channel) and
several hydrophobic interactions with other residues and the heme cofactor. 相似文献
16.
Peroxiredoxins (Prx) are a family of thiol dependent peroxidases found in almost all kingdoms. In plants, five major classes of Prx are known. They are known to catalyze the decomposition of peroxides and as they lack a prosthetic group, the catalytic cycle results in the generation of an inactive form of Prx. In order to regain the active form, Prx rely on external electron donors such as thioredoxins, glutaredoxins, cyclophilins, NADPH-dependent thioredoxin reductase C (NTRC) etc. In addition to their well established role in antioxidative defense, Prx are also reported to play an important role in growth and development, dessication tolerance in dormant seeds, protection of photosynthesis, defense against pathogens and redox signaling. Prx are also known to establish an alternate water–water cycle for the detoxification of H2O2, parallel to ascorbate-dependent H2O2 detoxification. But the relative contribution of Prx in detoxifying H2O2 compared to ascorbate peroxidase is not known so far due to experimental limitations. In view of the above, the present review focuses on the recent developments on Prxs. 相似文献
17.
Alexander V. Peskin Paul E. Pace Jessica B. Behring Louise N. Paton Marjolein Soethoudt Markus M. Bachschmid Christine C. Winterbourn 《The Journal of biological chemistry》2016,291(6):3053-3062
Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H2O2 and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m−1 s−1) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling. 相似文献
18.
Lian FM Yu J Ma XX Yu XJ Chen Y Zhou CZ 《The Journal of biological chemistry》2012,287(21):17077-17087
Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (CP) to Cys-62 and the resolving cysteine (CR) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ∼2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 Å resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing CP and CR. An intermolecular CP-CR disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32. 相似文献
19.
Emmanuel S. Burgos Carola Wilczek Takashi Onikubo Jeffrey B. Bonanno Janina Jansong Ulf Reimer David Shechter 《The Journal of biological chemistry》2015,290(15):9674-9689
The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site. 相似文献
20.
Yuji Masuda Miki Suzuki Hidehiko Kawai Asami Hishiki Hiroshi Hashimoto Chikahide Masutani Takashi Hishida Fumio Suzuki Kenji Kamiya 《Nucleic acids research》2012,40(20):10394-10407
Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6–RAD18 (an E2–E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2–UBC13 (a UEV–E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication. 相似文献