Glutathione transferase: new model for glutathione activation

Glutathione transferases are enzymes of the cellular detoxification system that metabolize a vast spectrum of xenobiotic and endobiotic toxic compounds. They are homodimers or heterodimers and each monomer has an active center composed of a G-site in which glutathione (GSH) binds and an H-site for the electrophilic substrate. When GSH binds to the G-site, the pKa value of its thiol group drops by 2.5 units; this promotes its deprotonation and, therefore, produces a strong nucleophilic thiolate that is able to react with the electrophilic substrate. The mechanism behind the deprotonation of the thiol group is still unknown. Some studies point to the fact that the GSH glutamyl alpha-carboxylate group is essential for GSH activation, whereas others indicate the importance of the active-center water molecules. On the basis of QM/MM calculations, we propose a mechanism of GSH activation in which a water molecule, acting as a bridge, is able to assist in the transfer of the proton from the GSH thiol group to the GSH glutamyl alpha-carboxylate group, after an initial GSH conformational rearrangement. We calculated the potential of mean force of this GSH structural rearrangement that would be necessary for the approach of both groups and we then performed a QM/MM ONIOM scan of water-assisted proton transfer. The overall free-energy barrier for the process is consistent with experimental studies of the enzyme kinetics.     

Substrate Profiling of Glutathione S‐transferase with Engineered Enzymes and Matched Glutathione Analogues

The identification of specific substrates of glutathione S‐transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G‐W40A‐R41A mutant (GST‐KWR) of the mu‐class glutathione S‐transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH‐R1) as the cosubstrate. The GSH‐R1 conjugation products can react with an azido‐based biotin probe for ready enrichment and MS identification. Proof‐of‐principle studies were carried to detect the products of GSH‐R1 conjugation to 1‐chloro‐2,4‐dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.     

Glutathione in Brain Disorders and Aging

Glutathione is a remarkably functional molecule with diverse features, which include being an antioxidant, a regulator of DNA synthesis and repair, a protector of thiol groups in proteins, a stabilizer of cell membranes, and a detoxifier of xenobiotics. Glutathione exists in two states—oxidized and reduced. Under normal physiological conditions of cellular homeostasis, glutathione remains primarily in its reduced form. However, many metabolic pathways involve oxidization of glutathione, resulting in an imbalance in cellular homeostasis. Impairment of glutathione function in the brain is linked to loss of neurons during the aging process or as the result of neurological diseases such as Huntington’s disease, Parkinson’s disease, stroke, and Alzheimer’s disease. The exact mechanisms through which glutathione regulates brain metabolism are not well understood. In this review, we will highlight the common signaling cascades that regulate glutathione in neurons and glia, its functions as a neuronal regulator in homeostasis and metabolism, and finally a mechanistic recapitulation of glutathione signaling. Together, these will put glutathione’s role in normal aging and neurological disorders development into perspective.     

Glutathione transferase A1-1: catalytic role of water

For more than almost 30 years now, glutathione transferases (GSTs) have been known as xenobiotic/endobiotic detoxification enzymes. GSTs catalyze the nucleophilic addition of glutathione (GSH) sulphur thiolate to a wide range of electrophilic substrates, building up a less toxic and more soluble compound, which can then be removed from the cell. Recently we proposed a consistent GSH activation mechanism. By performing QM/MM calculations, we demonstrated that a water molecule, following a first conformational rearrangement of GSH, is capable of assisting a proton transfer between the GSH thiol and alpha carboxylic groups. In this study we go further in the analysis of the water role in GSH activation by performing a long Molecular Dynamics (MD) study on glutathione transferase A1-1 Thr68 mutants complexed with GSH and the GSH decarboxylated analogue (dGSH), for which experimental kinetic data are available.     

电位法测定谷胱甘肽转移酶活性的研究

谷胱甘肽转移酶(GSTs)是生物体内一种重要的解毒酶,催化异源物与谷胱甘肽结合,有多种方法测定其活性,但都基于大分子产物。本实验基于H.Habig方法,探讨用氯离子选择电极,根据反应体系中Cl-浓度的变化来测定谷胱甘肽转移酶的活性。研究结果表明,利用透析膜包裹电极可以消除底物谷胱甘肽(GSH)对电极的干扰,生物反应体系中可能存在的离子、小分子(如Br-I、-、H2O2和Vc)对电极没有影响。此方法重现性良好,相对标准偏差为3.54%。     

Dithiophosphate-Induced Redox Conversions of Reduced and Oxidized Glutathione

Phosphorus species are potent modulators of physicochemical and bioactive properties of peptide compounds. O,O-diorganyl dithiophoshoric acids (DTP) form bioactive salts with nitrogen-containing biomolecules; however, their potential as a peptide modifier is poorly known. We synthesized amphiphilic ammonium salts of O,O-dimenthyl DTP with glutathione, a vital tripeptide with antioxidant, protective and regulatory functions. DTP moiety imparted radical scavenging activity to oxidized glutathione (GSSG), modulated the activity of reduced glutathione (GSH) and profoundly improved adsorption and electrooxidation of both glutathione salts on graphene oxide modified electrode. According to NMR spectroscopy and GC–MS, the dithiophosphates persisted against immediate dissociation in an aqueous solution accompanied by hydrolysis of DTP moiety into phosphoric acid, menthol and hydrogen sulfide as well as in situ thiol-disulfide conversions in peptide moieties due to the oxidation of GSH and reduction of GSSG. The thiol content available in dissolved GSH dithiophosphate was more stable during air oxidation compared with free GSH. GSH and the dithiophosphates, unlike DTP, caused a thiol-dependent reduction of MTS tetrazolium salt. The results for the first time suggest O,O-dimenthyl DTP as a redox modifier for glutathione, which releases hydrogen sulfide and induces biorelevant redox conversions of thiol/disulfide groups.     

Intrinsic Neutral and Anionic Structures of Glutathione

Gas‐phase intrinsic structures of intact neutral and anionic glutathione (GSH) have been determined by means of a combination of negative ion photo‐electron spectroscopy and quantum chemistry calculations. The inferred structures of the neutral parents of those peptide anions are canonical (non‐zwitterionic). These intrinsic structures are compared to those already known in aqueous solution or determined by crystallography in binding sites of enzymes.     

纳米电化学生物传感器

纳米电化学生物传感器是将纳米材料作为一种新型的生物传感介质,与特异性分子识别物质如酶、抗原/抗体、DNA等相结合,并以电化学信号为检测信号的分析器件。本文简要介绍了生物传感器的分类和纳米材料在电化学生物传感器中的应用及其优势,综述了近年来各类纳米电化学生物传感器在生物检测方面的研究进展,包括纳米颗粒生物传感器,纳米管、纳米棒、纳米纤维与纳米线生物传感器,以及纳米片与纳米阵列生物传感器等。     

A novel method for screening the glutathione transferase inhibitors

Background  

Glutathione transferases (GSTs) belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST) inhibitors, a novel method was designed.     

Smartphone-Assisted Colorimetric Detection of Glutathione and Glutathione Reductase Activity in Human Serum and Mouse Liver Using Hemin/G-Quadruplex DNAzyme

Abnormal levels of reduced glutathione (GSH) and glutathione reductase (GR) are usually related to a variety of diseases, so it is of great significance to determine the GSH concentration and GR activity. We herein develop a smartphone-assisted colorimetric biosensor for the detection of GSH and GR activity in human serum and mouse liver using hemin/G-quadruplex DNAzyme. Firstly, an obvious color change from colorless to green can be observed, owing to the high peroxidase-like activity of hemin/G-quadruplex DNAzyme toward 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS). With the addition of GSH or GR, the H₂O₂-mediated oxidation of ABTS catalyzed by hemin/G-quadruplex DNAzyme is significantly inhibited, resulting in remarkable color fading. Therefore, the detection of GSH and GR activity can be achieved by observing the color transition or measuring the absorbance at 420 nm. The detection limit was estimated to be as low as 0.1 μM and 10 μU/mL for GSH and GR, respectively. More interestingly, the RGB values of the sensing system can be identified by the smartphone application (APP, color collect), which makes it an ideal format for on-site determination and point-of-care testing (POCT). In addition, the proposed method shows excellent selectivity and acceptable applicability for the determination of GSH concentration and GR activity in human serum samples and mouse liver tissues, which might hold great application potential in clinical diagnosis and drug screening.     

Analyses of Antioxidative Properties of Selected Cyclitols and Their Mixtures with Flavanones and Glutathione

The conditions for determining the antioxidant properties of cyclitols (d-pinitol, l-quebrachitol, myo-, l-chiro-, and d-chiro-inositol), selected flavanones (hesperetin, naringenin, eriodictyol, and liquiritigenin) and glutathione by spectrophotometric methods—CUPRAC and with DPPH radical, and by a chromatographic method DPPH-UHPLC-UV, have been identified. Interactions of the tested compounds and their impact on the ox-red properties were investigated. The RSA (%) of the compounds tested was determined. Very low antioxidative properties of cyclitols, compared with flavanones and glutathione alone, were revealed. However, a significant increase in the determined antioxidative properties of glutathione by methyl-ether derivatives of cyclitols (d-pinitol and l-quebrachitol) was demonstrated for the first time. Thus, cyclitols seem to be a good candidate for creating drugs for the treatment of many diseases associated with reactive oxygen species (ROS) generation.     

Catalytic Reduction of Graphene Oxide Nanosheets by Glutathione Peroxidase Mimetics Reveals a New Structural Motif in Graphene Oxide

A catalytic reduction of graphene oxide (GO) by glutathione peroxidase (GPx) mimics is reported. This study reveals that GO contains peroxide functionalities, in addition to the epoxy, hydroxyl and carboxylic acid groups that have been identified earlier. It also is shown that GO acts as a peroxide substrate in the GPx‐like catalytic activity of organoselenium/tellurium compounds. The reaction of tellurol, generated from the corresponding ditelluride, reduces GO through the glutathione (GSH)‐mediated cleavage of the peroxide linkage. The mechanism of GO reduction by the tellurol in the presence of GSH involves the formation of a tellurenic acid and tellurenyl sulfide intermediates. Interestingly, the GPx mimics also catalyze the decarboxylation of the carboxylic acid functionality in GO at ambient conditions. Whereas the selenium/tellurium‐mediated catalytic reduction/decarboxylation of GO may find applications in bioremediation processes, this study suggests that the modification of GO by biologically relevant compounds such as redox proteins must be taken into account when using GO for biomedical applications because such modifications can alter the fundamental properties of GO.     

苯并异硒唑酮氨基酸衍生物的谷胱甘肽过氧化物酶样活性

采用ＮＡＤＰＨ／ＧＳＳＧ还原酶为指示剂的偶合法测定了两类共７种新型苯并异硒唑酮氨基酸衍生物的ＧＳＨ－Ｐｘ活性．结果表明，所有模拟物均显示较好的ＧＳＨ－Ｐｘ样活性，并且具有明显的构－效关系．     

Biosensors containing acetylcholinesterase and butyrylcholinesterase as recognition tools for detection of various compounds

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes expressed in the human body under physiological conditions. AChE is an important part of the cholinergic nerves where it hydrolyses neurotransmitter acetylcholine. Both cholinesterases are sensitive to inhibitors acting as neurotoxic compounds. In analytical applications, the enzymes can serve as a biorecognition element in biosensors as well as simple disposable sensors (dipsticks) and be used for assaying the neurotoxic compounds. In the present review, the mechanism of AChE and BChE inhibition by disparate compounds is explained and methods for assaying the enzymes activity are shown. Optical, electrochemical, and piezoelectric biosensors are described. Attention is also given to the application of sol-gel techniques and quantum dots in the biosensors’ construction. Examples of the biosensors are provided and the pros and cons are discussed.     

2-Fluoro-ATP as a versatile tool for 19F NMR-based activity screening

19F NMR-based methods have found utility in activity-based screening assays. However, because enzymes catalyze a diverse set of reactions, a large variety of fluorinated substrates would need to be identified to target each one separately. We have developed a more streamlined approach that is applicable to many enzymes that utilize ATP as a substrate. In this method, a fluorine-containing ATP analogue, 2-fluoro-ATP, is used to monitor the reaction. Applications are described for nicotinamide adenine dinucleotide synthetase and 3-phosphoinositide dependent kinase-1. Fragment screening results for the latter indicate that this technique can identify compounds that inhibit as well as activate reactions. The present results, together with previous biochemical studies from other laboratories, have shown that 2-fluoro-ATP can serve as a substrate for nine enzymes that are representative of three of the six enzyme subclasses, namely the transferases, hydrolases, and ligases. This suggests that 2-fluoro-ATP is suitable as a universal tool for screening ATP-requiring enzymes. Importantly, 2-fluoro-ATP has been determined to be a valid substrate for a variety of kinases, including both small molecule and protein kinases, suggesting that it may be useful for investigating the large number of pharmaceutically relevant kinases.     

Incorporation of Tellurocysteine into Glutathione Transferase Generates High Glutathione Peroxidase Efficiency

A rival to native peroxidase! An existing binding site for glutathione was combined with the catalytic residue tellurocysteine by using an auxotrophic expression system to create an engineered enzyme that functions as a glutathione peroxidase from the scaffold of a glutathione transferase (see picture). The catalytic activity of the telluroenzyme in the reduction of hydroperoxides by glutathione is comparable to that of native glutathione peroxidase.

     

Antioxidant Effect of Selenium-containing Glutathione S-Transferase in Rat Cardiomyocytes

As one of the most important antioxidant enzymes, glutathione peroxidase(GPX) protects cells and tissues from oxidative damage, and plays an important role in cardiovascular and cerebrovascular injuries induced by oxidative stress. The antioxidant effect of selenium-containing glutathione S-transferase(Se-GST), a mimic of GPX was investigated on rat cardiomyocytes. To explore the protection function of Se-GST in hydrogen peroxide(H₂O₂) challenged rat cardiomyocytes, we examined malondialdehyde(MDA), lactate dehydrogenase(LDH), superoxide dismutase( SOD) and cell apoptosis. The results demonstrate exposure of rat cardiomyocytes to H₂O₂ for 6 and 12 h induced the significant increases of MDA, LDH and apoptosis rate of cardiomyocytes, but pretreatment of rat cardiomyocytes with Se-GST at 0.0005 or 0.001 unit/mL prevents oxidative stress induced by H₂O₂ with the decreases of cell apoptosis. All the results hint Se-GST has antioxidant activity for oxidative stress challenged rat cardiomyocytes.     

生物酶振荡器研究谷胱甘肽的抗氧化效应

组装了一种生物酶振荡器（Peroxidase-Oxidase Biochemical Oscillator） 的实验装置、建立了相应的实验方法,在此基础上得到单倍周期、四倍周期、混沌 等丰富的振荡图谱信息,并采用它作为“探针”,检测了谷胱甘肽（GSH）的抗氧 化效应,它可作为定量分析的基础,线性范围为9.38 * 10~(-7) ～ 7.50 * 10~(- 5) mol·L~(-1),探讨了某些可能的反应机理。     

Theoretical Study on GSH Activation Mechanism of a New Type of Glutathione Transferase Gtt2

Glutathione transferases(GSTs) play an important role in the detoxification of xenobiotic/endobiotic toxic compounds. The α-, π-, and μ-classes of cytosolic GSTs have been studied extensively, while Gtt2 from Saccharomyces cerevisiae, a novel atypical GST, is still poorly understood. In the present study, we investigated the glutathione( GSH) activation mechanism of Gtt2 using the density functional theory(DFT) with the hybrid functional B3LYP. The computational results show that a water molecule could assist a proton transfer between the GSH thiol and the N atom of His133. The energy barrier of proton transfer is 46.0 kJ/mol. The GSH activation mechanism and the characteristics of active site are different from those of classic cytosolic GSTs.     

Glutathione Amperometric Enzyme Microsensor

Abstract

Glutathione micro-enzyme sensors were developed based on the immobilization of glutathione oxidase at the tip of a 25 μm Pt wire sealed in glass. An inner membrane constructed from cellulose acetate (CA) was deposited onto the tip of the Pt microelectrode prior to enzyme immobilization with glutaraldehyde. The final outer diameter of the microelectrodes is approximately 30 μm. The analytical characteristics of the microelectrodes, including calibration curves, apparent K_M′, pH response curves, stability and selectivity over enzymatic interferences were determined. The response was linear in the concentration range 1.0 × 10^?5 M - 1.9 × 10^?4 M glutathione, reaching 95% steady-state current in 15–20 seconds. The microelectrodes were useful for more than two months.