共查询到20条相似文献,搜索用时 343 毫秒
1.
Chen Chen Qihong Sun Buvaneswari Narayanan Donald L. Nuss Osnat Herzberg 《The Journal of biological chemistry》2010,285(34):26685-26696
Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with ∼1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 Å, respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (α/β)8 barrel fold and each pair swapping the 8th α-helix. An active site “gating loop” exhibits conformational disorder in the ligand-free structure. To obtain the structures of the OAH·ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference. 相似文献
2.
Michelle Oppenheimer Todd L. Lowary Pablo Sobrado 《Archives of biochemistry and biophysics》2010,502(1):31-556
UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose, which is an important cell wall component in Aspergillus fumigatus and other pathogenic microbes. A. fumigatus UGM (AfUGM) was expressed in Escherichia coli and purified to homogeneity. The enzyme was shown to function as a homotetramer by size-exclusion chromatography and to contain ∼50% of the flavin in the active reduced form. A kcat value of 72 ± 4 s−1 and a KM value of 110 ± 15 μM were determined with UDP-galactofuranose as substrate. In the oxidized state, AfUGM does not bind UDP-galactopyranose, while UDP and UDP-glucose bind with Kd values of 33 ± 9 μM and 90 ± 30 μM, respectively. Functional and structural differences between the bacterial and eukaryotic UGMs are discussed. 相似文献
3.
Mesaconate is an intermediate in the glutamate degradation pathway of microorganisms such as Clostridium tetanomorphum. However, metabolic engineering to produce mesaconate has not been reported previously. In this work, two enzymes involved in mesaconate production, glutamate mutase and 3-methylaspartate ammonia lyase from C. tetanomorphum, were recombinantly expressed in Escherichia coli. To improve mesaconate production, reactivatase of glutamate mutase was discovered and adenosylcobalamin availability was increased. In addition, glutamate mutase was engineered to improve the in vivo activity. These efforts led to efficient mesaconate production at a titer of 7.81 g/L in shake flask with glutamate feeding. Then a full biosynthetic pathway was constructed to produce mesaconate at a titer of 6.96 g/L directly from glucose. In summary, we have engineered an efficient system in E. coli for the biosynthesis of mesaconate. 相似文献
4.
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
5.
With site-directed mutagenesis, Ser319 and Ser321 in
conserved stretch 3 of tetrameric isocitrate lyase from Escherichia
coli were each substituted with alanine, cysteine, asparagine, or
threonine in addition to simultaneous alanine/alanine substitutions. Besides
their absolute conservation in all aligned isocitrate lyase sequences, the
location of these serine residues, which flank a completely conserved
proline, had been suggested in the active site in previous research by
studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J
Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine
appreciably reduced the kcat of E. coli isocitrate lyase
relative to that for wild-type (100) as follows: S319A, 0.4; S319C, 0.05;
S319N, 0.01; S319T, 32.3; S321A, 2.9; S321C, 0.3; S321N, 0.1; S321T, 0.3; and
S319A/S321A, 0, with little or no effect on the K
m
for the
substrate Mg2+-Ds-isocitrate. The most active variant
S319T exhibited threefold less activity than the wild-type enzyme; all
variants assembled into tetramers. The S319T mutant isocitrate lyase was
100-fold more active than the S321T variant. This observation suggests that
the requirement for a β-hydroxymethyl group of serine in catalysis is less
important at position 319 than at position 321. Although most singly
substituted variants had very low isocitrate lyase activity, all variants
harboring mutant isocitrate lyase of very low activity did grow on acetate as
a sole carbon source albeit with longer doubling times and lag phases.
Substitution of Pro320 by Ala, Asp, Gly, or His was highly detrimental to
activity and increased the K
m
for substrate 3.5- to
8-fold; this suggests that Pro fixes the location of adjacent Ser OH groups
and facilitates substrate binding and catalysis. From these collective
results, it is proposed that Ser319 and Ser321 are involved in E.
coli isocitrate lyase catalysis, perhaps by stabilizing the postulated
reaction intermediate succinate trianion in the aci-carboxylate form
and the related transition state via hydrogen bonding.
Received: 3 September 1996 / Accepted: 20 September 1996 相似文献
6.
Biochemical studies to elucidate the structural basis for xyloglucan specificity among GH12 xyloglucanases are lacking. Accordingly, the substrate specificity of a GH12 xyloglucanase from Aspergillus niger (AnXEG12A) was investigated using pea xyloglucan and 12 xylogluco-oligosaccharides, and data were compared to a structural model of the enzyme. The specific activity of AnXEG12A with pea xyloglucan was 113 μmol min−1 mg−1, and apparent kcat and Km values were 49 s−1 and 0.54 mg mL−1, respectively. These values are similar to previously published results using xyloglucan from tamarind seed, and suggest that substrate fucosylation does not affect the specific activity of this enzyme. AnXEG12A preferred xylogluco-oligosaccharides containing more than six glucose units, and with xylose substitution at the −3 and +1 subsites. The specific activities of AnXEG12A on 100 μM XXXGXXXG and 100 μM XLLGXLLG were 60 ± 4 and 72 ± 9 μmol min−1 mg−1, respectively. AnXEG12A did not hydrolyze XXXXXXXG, consistent with other data that demonstrate the requirement for an unbranched glucose residue for hydrolysis by this enzyme. 相似文献
7.
Wenfang Dou Dan Wei Hui Li Heng Li Muhammad Masfiqur Rahman Jinsong Shi Zhenghong Xu Yanhe Ma 《Carbohydrate polymers》2013
A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0–8.0) and temperatures below 50 °C. Metal ions including Na+, Mg2+, Mn2+, and Ca2+ notably increased the activity of the enzyme. With sodium alginate as the substrate, the Km and Vmax were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees. 相似文献
8.
《Bioorganic & medicinal chemistry》2014,22(21):5961-5969
Antibiotic resistance is a growing health concern, and new avenues of antimicrobial drug design are being actively sought. One suggested pathway to be targeted for inhibitor design is that of iron scavenging through siderophores. Here we present a high throughput screen to the isochorismate-pyruvate lyase of Pseudomonas aeruginosa, an enzyme required for the production of the siderophore pyochelin. Compounds identified in the screen are high nanomolar to low micromolar inhibitors of the enzyme and produce growth inhibition in PAO1 P. aeruginosa in the millimolar range under iron-limiting conditions. The identified compounds were also tested for enzymatic inhibition of Escherichia coli chorismate mutase, a protein of similar fold and similar chemistry, and of Yersinia enterocolitica salicylate synthase, a protein of differing fold but catalyzing the same lyase reaction. In both cases, subsets of the inhibitors from the screen were found to be inhibitory to enzymatic activity (mutase or synthase) in the micromolar range and capable of growth inhibition in their respective organisms (E. coli or Y. enterocolitica). 相似文献
9.
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
10.
Takayoshi Tagami Masayuki Okuyama Hiroyuki Nakai Young-Min Kim Haruhide Mori Kazunori Taguchi Birte Svensson Atsuo Kimura 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):329-335
Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest kcat/Km for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased kcat/Km values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the kcat/Km values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest kcat/Km value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at + 2 and + 3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs. 相似文献
11.
Chiara Ciaccio Grazia R. Tundo Giuseppe Grasso Daniela Marasco Magda Gioia Massimo Coletta 《Journal of molecular biology》2009,385(5):1556-1567
Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [kcat (s− 1) = 0.38 (± 0.05); Km (M) = 7.5 (± 0.9) × 10− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the Km toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn2+ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain. 相似文献
12.
Aspergillus niger produces oxalic acid through the hydrolysis of oxaloacetate, catalyzed by the cytoplasmic enzyme oxaloacetate acetylhydrolase (OAH). The A. niger genome encodes four additional open reading frames with strong sequence similarity to OAH yet only the oahA gene encodes OAH activity. OAH and OAH-like proteins form subclass of the isocitrate lyase/PEP mutase enzyme superfamily, which is ubiquitous present filamentous fungi. Analysis of function-specific residues using a superfamily-based approach revealed an active site serine as a possible sequence marker for OAH activity. We propose that presence of this serine in family members correlates with presence of OAH activity whereas its absence correlates with absence of OAH. This hypothesis was tested by carrying out a serine mutagenesis study with the OAH from the fungal oxalic acid producer Botrytis cinerea and the OAH active plant petal death protein as test systems. 相似文献
13.
Francisco J. FernandezDominique de Vries Esther Peña-Soler Miquel CollPhilipp Christen Heinz GehringM. Cristina Vega 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(2):339-349
The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 105-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, kcat′ for desulfination of l-cysteine sulfinate increased to 0.5 s− 1 (from 0.05 s− 1 in wild-type enzyme), whereas kcat′ for transamination of the same substrate was reduced from 510 s− 1 to 0.05 s− 1. Similarly, kcat′ for β-decarboxylation of l-aspartate increased from < 0.0001 s− 1 to 0.07 s− 1, whereas kcat′ for transamination was reduced from 530 s− 1 to 0.13 s− 1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water. 相似文献
14.
Saccharomyces cerevisiae Atm1p has been cloned, over-expressed and purified from a yeast expression system. The sequence includes both the soluble ATPase and transmembrane-spanning domains. With the introduction of an N-terminal Kozak sequence and a C-terminal (His)6-tag, a yield of 1 mg of Atm1p was obtained from 3 g wet yeast cells, which is comparable to other membrane-associated proteins isolated from eukaryotic expression systems. The ATPase activity of Atm1p is sensitive to sodium vanadate, a P-type ATPase inhibitor, with an IC50 of 4 μM. MgADP is a product inhibitor for Atm1p with an IC50 of 0.9 mM. The Michaelis–Menten constants Vmax, KM and kcat of Atm1p were measured as 8.7 ± 0.3 μM/min, 107 ± 16 μM and 1.24 ± 0.06 min− 1, respectively. A plot of ATPase activity versus concentration of Atm1p exhibits a nonlinear relationship, suggesting an allosteric response and an important role for the transmembrane domain in mediating both ATP hydrolysis and MgADP release. The metal dependence of Atm1p ATPase activity demonstrated a reactivity order of Mg2+ > Mn2+ > Co2+, while each divalent ion was found to be inhibitory at higher concentrations. The activation and inhibitory effect of phospholipids suggest that formation of a lipid–micelle complex is important for enzymatic activity and stability. Structural analysis of Atm1p by CD spectroscopy suggested a similarity of secondary structure to that found for other members of this ABC protein family. 相似文献
15.
Synthesis of Isocitrate Lyase in Sunflower Cotyledons during the Transition in Cotyledonary Microbody Function 总被引:3,自引:3,他引:0 下载免费PDF全文
Density-labeling with 10 millimolar K15NO3/70% 2H2O has been used to investigate isocitrate lyase synthesis during greening of sunflower (Helianthus annuus L.) cotyledons when the glyoxysomal enzyme activities sharply decline and the transition in cotyledonary microbody function occurs. A density shift of 0.0054 (kilograms per liter) was obtained for the profile of isocitrate lyase activity in the CsCl gradient with respect to the 1H2O control. Quantitative evaluation of the density-labeling data indicates that about 50% of the isocitrate lyase activity present towards the end of the transition stage in microbody function is due to enzyme molecules newly synthesized during this stage. 相似文献
16.
Divergent Metabolic Pathways for Propane and Propionate Utilization by a Soil Isolate 总被引:14,自引:9,他引:5 下载免费PDF全文
The metabolism of propane and propionate by a soil isolate (Brevibacterium sp. strain JOB5) was investigated. The presence of isocitrate lyase in cells grown on isopropanol, acetate, or propane and the absence of this inducible enzyme in n-propanol- and propionate-grown cells suggested that propane is not metabolized via C-terminal oxidation. Methylmalonyl coenzyme A mutase and malate synthase are constitutive in this organism. The incorporation of 14CO2 into pyruvate accumulated during propionate utilization suggests that propionate is metabolized via the methyl-malonyl-succinate pathway. These results were further substantiated by radiorespirometric studies with propionate-1-14C, -2-14C, and -3-14C as substrate. Propane -2-14C was shown, by unlabeled competitor experiments, to be oxidized to acetone; acetone and isopropanol are oxidized in this organism to acetol. Cleavage of acetol to acetate and CO2 would yield the inducer for the isocitrate lyase present in propane-grown cells. 相似文献
17.
Ryuichiro Suzuki Kazue Terasawa Keitarou Kimura Zui Fujimoto Mitsuru Momma Mikihiko Kobayashi Atsuo Kimura Kazumi Funane 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(7):919-924
Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the kcat/KM value of CITase-598K was approximately two times higher (32.2 s− 1 mM− 1) than that of CITase-T3040 (17.8 s− 1 mM− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes. 相似文献
18.
The succinate analog itaconic acid was observed to be a competitive inhibitor of the glyoxylate cycle specific enzyme isocitrate lyase (EC 4.1.3.1) in cell-free extracts of Tetrahymena pyriformis. Itaconic acid also inhibited net in vivo glycogen synthesis from glyoxylate cycle-dependent precursors such as acetate but not from glyoxylate cycle-independent precursors such as fructose. The effect of itaconic acid on the incorporation of 14C into glycogen from various 14C-labeled precursors was also consistent with inhibition of isocitrate lyase by this compound. Another analog of succinate which shares a common metabolic fate with itaconic acid, mesaconic acid, had no effect on isocitrate lyase activity in vitro or on 14C-labeled precursor incorporation into glycogen in vivo. In addition, itaconic acid did not affect gluconeogenesis from lactate in isolated perfused rat livers, a system lacking the enzyme isocitrate lyase. These results are taken as evidence that itaconic acid is an inhibitor of glyoxylate cycle-dependent glyconeogenesis Tetrahymena pyriformis via specific competitive inhibition of isocitrate lyase activity. 相似文献
19.
Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N2O (2NO + 2e− + 2H+ → N2O + H2O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O2-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O2, we demonstrate that protons are indeed consumed from the ‘outside’. First, multiple turnover reduction of O2 resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O2 shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O2 as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O2 show electron transfer coupled to proton uptake from outside the NOR-liposomes with a τ = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Ädelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested. 相似文献
20.
Francesco Sgarrella Luciano Frassetto Simone Allegrini Marcella Camici Maria Caterina Carta Paolo Fadda Maria Grazia Tozzi Piero Luigi Ipata 《Biochimica et Biophysica Acta (BBA)/General Subjects》2007
Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N6-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164 ± 5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (kcat/Km 2.1 × 106 s− 1 M− 1), followed by 2′-deoxyadenosine (kcat/Km 4.2 × 105 s− 1 M− 1). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding. 相似文献