Protein conformational gating of enzymatic activity in xanthine oxidoreductase

In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E(sq/hq)) is ~170 mV, a striking difference. The former greatly prefers NAD(+) as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD(+)), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 ? resolution crystal structures for XDH, XO, the NAD(+)- and NADH-complexed XDH, E(sq/hq) were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E(sq/hq) difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD(+) binding at XDH. Instead, the positive charge of the NAD(+) ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD(+) molecule all contribute to altering E(sq/hq) upon NAD(+) binding to XDH.     

Oxidase-cofactor electrodes aimed at the quantitative measure of substrate concentrations

Two approaches are described briefly for utilizing oxidase enzymes in electrochemical sensors. In one approach an oxidase enzyme flavin cofactor was covalently coupled to the electrode surface with the anticipated readout being a current proportional to the concentration of enzyme substrate. In the second approach the enzyme glucose oxidase was immobilized on platinum such that the potential of the platinum varied with the concentration of glucose in a solution buffered at pH 7.4. The status of the two projects is described.     

514— Oxidase-cofactor electrodes aimed at the quantitative measure of substrate concentrations

Two approaches are described briefly for utilizing oxidase enzymes in electrochemical sensors. In one approach an oxidase enzyme flavin cofactor was covalently coupled to the electrode surface with the anticipated readout being a current proportional to the concentration of enzyme substrate. In the second approach enzyme glocose oxidase was immobilized on platinum such that the potential of the platinum varied with the concentration of glucose in a solution buffered at pH 7.4. The status of the two projects is described.     

Surface reconstitution of glucose oxidase onto a norbornylogous bridge self-assembled monolayer

An electrode construct was fabricated in which a self-assembled monolayer containing a novel norbornylogous bridge was covalently attached to flavin adenine dinucleotide (FAD), the redox active centre of several oxidase enzymes. The electrochemistry of the construct was investigated before and after the reconstitution of glucose oxidase around the surface bound FAD. Rapid rates of electron transfer were observed both before and after the reconstitution of biocatalytically active enzyme. However, no biocatalytic activity was observed under anaerobic conditions suggesting the a lack of enzyme turnover through direct electron transfer. It is proposed that a decrease in the electronic coupling between the redox active FAD and the electrode following reconstitution of the glucose oxidase – a probable consequence of the FAD being immersed in a protein environment – was responsible for the inability of the enzyme to be turned over under anaerobic conditions.     

Modeling the surface phenomena in carbon paste electrodes by low frequency impedance and double-layer capacitance measurements

Impedance and capacitance studies have been performed with covalently coupled Glucose oxidase (GOD) enzyme, covalently coupled flavin adenine dinucleotide (FAD), reconstituted GOD enzyme and blank carbon paste electrodes to study the changes in the electrochemical interfacial properties. Impedance studies were performed using a low frequency impedance technique and the electrochemical surface capacitance was measured by a pulse technique. We have attempted to fit the experimental values to an equivalent circuit model. The Randles' cell circuit with Warburg impedance modeled well the experimental values and the behavior of the enzyme electrodes. The individual components of the model were calculated and the parameters were explained. The blank paste electrode showed a constant phase element behavior.     

Characterization of the covalently bound anionic flavin radical in monoamine oxidase a by electron paramagnetic resonance

It was recently suggested that partially reduced monoamine oxidase (MAO) A contains an equilibrium mixture of an anionic flavin radical and a tyrosyl radical (Rigby, S. E.; et al. J. Biol. Chem. 2005, 280, 4627-4632). These observations formed the basis for a revised radical mechanism for MAO. In contrast, an earlier study of MAO B only found evidence for an anionic flavin radical (DeRose, V. J.; et al. Biochemistry 1996, 35, 11085-11091). To resolve the discrepancy, we have performed continuous-wave electron paramagnetic resonance at 94 GHz (W-band) on the radical form of MAO A. A comparison with d-amino acid oxidase (DAAO) demonstrates that both enzymes only contain anionic flavin radicals. Pulsed electron-nuclear double resonance spectra of the two enzymes recorded at 9 GHz (X-band) reveal distinct hyperfine coupling patterns for the two flavins. Density functional theory calculations show that these differences can be understood in terms of the difference at C8alpha of the isoalloxazine ring. DAAO contains a noncovalently bound flavin whereas MAO A contains a flavin covalently bound to a cysteinyl residue at C8alpha. The similar electronic structures and hydrophobic environments of MAO and DAAO, and the similar structural motifs of their substrates suggest that a direct hydride transfer catalytic mechanism established for DAAO (Umhau, S.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12463-12468) should be considered for MAO.     

Achieving Direct Electrical Connection to Glucose Oxidase Using Aligned Single Walled Carbon Nanotube Arrays

Direct electron transfer between an electrode and the redox active centre of glucose oxidase, flavin adenine dinucleotide (FAD), is probed using carbon nanotube modified gold electrodes. Gold electrodes are first modified with a self‐assembled monolayer of cysteamine and then shortened single walled carbon nanotubes (SWNT) are aligned normal to the electrode surface by self‐assembly. The electrochemistry of these aligned nanotube electrode arrays is initially investigated using potassium ferricyanide which showed SWNT act as nanoelectrodes with the ends of the tubes more electrochemically active than the walls. Subsequently the nanotubes are plugged into the enzymes in one of two ways. In the first method, native glucose oxidase is covalently attached to the ends of the aligned tubes which allowed close approach to FAD and direct electron transfer to be observed with a rate constant of 0.3 s^?1. In the second strategy, FAD was attached to the ends of the tubes and the enzyme reconstituted around the surface immobilized FAD. This latter approach allowed more efficient electron transfer to the FAD with a rate constant of 9 s^?1.     

Artificial redox enzymes. II. A computational chemistry study1

A computational chemistry study of the artificial redox enzyme synthesized by covalently attaching flavin to cyclodextrins explains some of its properties. Calculations indicate that the flavin moiety covalently attached to cyclodextrin is not within the cavity of cyclodextrin. This result is consistent with the UV-vis spectrum of the artificial enzyme. The calculations also indicate hydrogen bonds formed between the carbonyl groups of the catalytic functionality and the hydroxyl groups of cyclodextrin play a role in their most stable conformation. This explains the observed overall stability of these artificial enzymes compared to riboflavin. Electrostatic energies and solvation energies play a major role in the stability of the hosts and the orientation of guests included within the artificial enzymes. The rates of oxidation of various thiols catalyzed by the artificial enzyme can be explained by the relative distances between the sulfur atom of the substrates and C(4a) of the flavin moiety.     

Reductive metabolism of nitro-p-phenylenediamine by rat liver

Reductive metabolism of the hair dye constituent, nitro-p-phenylenediamine (2-nitro-1,4-diaminobenzene, NPDA), and its acetylated metabolite, NPDA N4-acetate, was investigated with rat liver subcellular fractions, microsomes and cytosol. Under anaerobic conditions, these compounds were reduced to their corresponding amines by these fractions. The microsomal nitro-reducing activity was retarded completely by air and strongly by carbon monoxide. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) functioned more effectively than reduced nicotinamide adenine dinucleotide (NADH) as an electron donor in the microsomal reduction of the nitro compounds, and flavin mononucleotide (FMN) gave rise to a marked enhancement in the microsomal activity, especially when added to an anaerobic incubation mixture containing both NADH and NADPH. The cytosolic nitro-reducing activity was attributed to xanthine oxidase, aldehyde oxidase and other unknown enzyme(s), based on the results of cofactor requirements and inhibition experiments.     

离心超滤质谱法筛选中药复方二妙丸中黄嘌呤氧化酶抑制剂

采用离心超滤质谱分析技术(UF-UPLC/Q-TOF/MS), 结合体外酶活性实验方法, 对中药复方二妙丸提取物中的黄嘌呤氧化酶抑制剂进行了筛选. 体外酶活性实验测得二妙丸水提液对黄嘌呤氧化酶的半数抑制浓度(IC₅₀)为(0.218±0.0034) mg/mL, 表明二妙丸具有较强的黄嘌呤氧化酶抑制活性. 进一步采用离心超滤质谱技术对二妙丸水提物中潜在的黄嘌呤氧化酶抑制剂进行筛选, 从中筛选并鉴定了9种具有潜在黄嘌呤氧化酶抑制活性的化合物, 为开发黄嘌呤氧化酶抑制剂及阐明二妙丸治疗痛风和高尿酸血症的作用机制提供了一定的依据.     

Biomimetics with a self-assembled monolayer of catalytically active tethered isoalloxazine on Au

A new biomimetic nanostructured electrocatalyst comprised of a self-assembled monolayer (SAM) of flavin covalently attached to Au by reaction of methylformylisoalloxazine with chemisorbed cysteamine is introduced. Examinations by Fourier transform infrared spectroscopy and scanning tunneling microscopy (STM) show that the flavin molecules are oriented perpendicular to the surface with a 2 nm separation between flavin molecules. As a result of the contrast observed in the STM profiles between areas only covered by unreacted cysteamine and those covered by flavin-cysteamine moieties, it can be seen that the flavin molecules rise 0.7 nm above the chemisorbed cysteamines. The SAM flavin electrocatalyst undergoes fast electron transfer with the underlying Au and shows activity toward the oxidation of enzymatically active beta-NADH at pH 7 and very low potential (-0.2 V vs Ag/AgCl), a requirement for use in an enzymatic biofuel cell, and a 100-fold increase in activity with respect to the collisional reaction in solution.     

聚苯胺黄嘌呤氧化酶电极的生物电化学活性

用电化学方法将黄嘌呤氧化酶固定在聚苯胺中以制成聚苯胺黄嘌呤氧化酶电极。该电极呈现典型的酶催化反应动力学特性。且具有快速的生物电化学响应。固定化黄嘌呤氧化酶的表观米氏常数为21×10^-^6mol.dm^-^3, 最适pH为8.4,酶催化反应的活化能为85.1kJ.mol^-^1。酶电极具有较高的稳定性。使用聚苯胺黄嘌呤氧化酶电极能可靠地测定较低的底物浓度, 如2×10^-^6mol.dm^-^3黄嘌呤。     

TRIPLET STATE STUDIES OF FLAVINS BY ELECTRON PARAMAGNETIC RESONANCE—I

Abstract— The triplet state of flavin derivatives and d-amino acid oxidase was observed by electron paramagnetic resonance at 77°K.
Flavin triplets (Δ m =± 2) originate from the isoalloxazine ring and are resonant at 1560 guass.The half-life of the FMN triplet in 1 N HCl is 15 nisec.This life-time is prolonged indirectly by the presence of paramagnetic species, such as oxygen or free radicals.
The flavin triplet state is pH dependent.In neutral solution the nlaximum triplet yield is obtained and the longest life-time is observed.The triplet state is affected by intra-and inter-molecular complex formation, FAD is partially quenched by indirectly substituted adenine.Tryptophan quenches completely the FMN triplet.The FAD triplet of d-amino acid oxidase is enhanced but the life-time is shortened relative to a pure FAD solution.     

Homogeneous immunoassay for the detection of trinitrotoluene (TNT) based on the reactivation of apoglucose oxidase using a novel FAD-trinitrotoluene conjugate

A flavin adenine dinucleotide-trinitrotoluene derivative (FAD-TNT) was synthesized by coupling N ⁶-(2-aminoethyl)-FAD covalently to the N-hydroxysuccinimidyl ester of trinitrophenyl-γ-aminobutyric acid and characterized by negative-ion electrospray-ionization mass spectrometry (ESI-MS) after purification by reversed-phase HPLC. Free FAD-TNT can be detected at very low levels by recombination with apoglucose oxidase, since the FAD-TNT-glucose oxidase complex is enzymatically active. On the contrary, if FAD-TNT has been bound by an anti-TNT antibody, the conjugate cannot recombine with apoglucose oxidase any more. Based on these two phenomena, a homogeneous apoenzyme reactivation immunoassay system (ARIS) was developed for the detection of TNT. No separation step is needed in this assay. Proportionality between the TNT concentration and enzyme activity was demonstrated with a detection limit of 5 μg/L TNT.     

Screening,separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi‐mode microplate reader

Natural products have become one of the most important resources for discovering novel xanthine oxidase inhibitors, which are commonly employed in the treatment of hyperuricemia and gout. However, to date, few reports exist regarding the use of monoterpene glycosides as xanthine oxidase inhibitors. Thus, we herein report the use of ultrafiltration coupled with liquid chromatography in the screening of monoterpene glycoside xanthine oxidase inhibitors from the extract of Paeonia lactiflora (P. lactiflora ), and both high‐performance counter‐current chromatography and medium‐pressure liquid chromatography were employed to separate the main constituents. Furthermore, the xanthine oxidase inhibitory activities and the mechanisms of inhibition of the isolated compounds were evaluated using a multi‐mode microplate reader by Molecular Devices. As a result, three monoterpene glycosides were separated by combined high‐performance counter‐current chromatography and medium‐pressure liquid chromatography in purities of 90.4, 98.0, and 86.3%, as determined by liquid chromatography. These three compounds were identified as albiflorin, paeoniflorin, and 1‐O‐β‐ᴅ‐glucopyranosyl‐8‐O‐benzoylpaeonisuffrone by electrospray ionization tandem mass spectrometry, and albiflorin and paeoniflorin were screened as potential xanthine oxidase inhibitors by ultrafiltration with liquid chromatography. The evaluation results of xanthine oxidase inhibitory activity corresponded with the screening results, as only albiflorin and paeoniflorin exhibited xanthine oxidase inhibitory activity.     

Mimicking primary processes in photosynthesis—covalently linked porphyrin quinones

Porphyrin quinones (P-Qs), covalently linked via different aliphatic bridges, have been synthesized and studies in their (porphyrin) cationic and (semiquinone) anionic radical states by EPR, ENDOR and TRIPLE resonance techniques. Electron transfer (ET) from the porphyrin donor to the quinone acceptor could be observed by time-resolved picosecond fluorescence spectroscopy (singlet ET) and by time-resolved EPR spectroscopy (triplet ET) in isotropic fluid solution and in anisotropic media (liquid crystals and reversed micelles). Steady-state in situ photoexcitation of P-Qs in CTAB cationic reversed micelles yielded the corresponding semiquinone radical anions. In TRITON X-100 reversed micelles both the radical cation of the porphyrin and the radical anion of the semiquinone could be detected, which occured in complete emission. In covalently linked porphyrin flavins ET from the photoexcited porphyrin fragment to the flavin and, in addition, energy transfer from the photoexcited flavin to the porphyrin could be observed.     

Photoreactivation of alloxanthine-inhibited xanthine oxidase

Alloxanthine-inhibited xanthine oxidase (XOD) was found to be photoreactivated by irradiation of light of wavelengths in the range of 340-430 nm. The enzyme activity can be fully controlled to be on or off by many dark-light cycles. Electron spin resonance measurement shows the appearance of the molybdenum (V) ion and the reduced form of flavin adenine dinucleotide (FADH.) radical signals after irradiation of the alloxanthine-XOD complex. Electronic-absorption spectrum also shows the bleaching of Fe/S and flavin adenine dinucleotide chromophores at 375 and 450 nm as well as broad-band absorption of FADH. in the range of 500-700 nm. The quantum yield of photoreactivation of the enzyme activity is approximately 0.06. A photoinduced intraenzyme electron-transfer model is proposed to rationalize the photoreactivation process.     

用聚苯胺膜从牛乳中直接分离黄嘌呤氧化酶

酶电极;新鲜牛乳;固定分离;用聚苯胺膜从牛乳中直接分离黄嘌呤氧化酶     

Homogeneous immunoassay for the detection of trinitrotoluene (TNT) based on the reactivation of apoglucose oxidase using a novel FAD-trinitrotoluene conjugate

A flavin adenine dinucleotide-trinitrotoluene derivative (FAD-TNT) was synthesized by coupling N ⁶-(2-aminoethyl)-FAD covalently to the N-hydroxysuccinimidyl ester of trinitrophenyl-γ-aminobutyric acid and characterized by negative-ion electrospray-ionization mass spectrometry (ESI-MS) after purification by reversed-phase HPLC. Free FAD-TNT can be detected at very low levels by recombination with apoglucose oxidase, since the FAD-TNT-glucose oxidase complex is enzymatically active. On the contrary, if FAD-TNT has been bound by an anti-TNT antibody, the conjugate cannot recombine with apoglucose oxidase any more. Based on these two phenomena, a homogeneous apoenzyme reactivation immunoassay system (ARIS) was developed for the detection of TNT. No separation step is needed in this assay. Proportionality between the TNT concentration and enzyme activity was demonstrated with a detection limit of 5 μg/L TNT. Received: 5 September 1997 / Revised: 3 December 1997 / Accepted: 9 December 1997     

Simultaneous assay of hypoxanthine, xanthine and allopurinol by high-performance liquid chromatography and activation of immobilized xanthine oxidase as an enzyme reactor

A selective and sensitive assay of substrates (hypoxanthine, xanthine and allopurinol) of xanthine oxidase by reversed-phase liquid chromatography coupled with the use of immobilized enzyme reactors is described. These compounds were oxidized by immobilized xanthine oxidase and produced hydrogen peroxide, which was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. The detection limits of hypoxanthine, xanthine and allopurinol were approximately 50, 120 and 130 pg per injection, respectively. Immobilized xanthine oxidase inhibited by oxipurinol during the assay was reactivated by 2,6-dichlorophenolindophenol and could be used for a long period without a significant activity loss. These methods were applied to plasma and urine samples.