Thiol Enzyme Research Articles

Effect of Methyl Jasmonate on the Gene Expression, Encoding Non-Protein Thiol Enzymes in Wheat under Cadmium.

The effect of the phytohormone methyl jasmonate (MJ) on the expression of the TaGS1 and TaPCS1 genes encoding glutathione synthetase and phytochelatin synthase, respectively, which are key enzymes in the synthesis of glutathione and phytochelatins in wheat (cv. Moskovskaya 39), was studied. It was shown for the first time that pretreatment of plants by exogenous MJ (1 μM) leads to an increase in the accumulation of transcripts of the TaGS1 and TaPCS1 genes in leaves without cadmium effect. When cadmium (CdSO4, 100 μM) was added into the nutrient solution, the transcript level of the TaGS1 gene in the plants pretreated by MJ increased (compared to the untreated plants), whereas the transcript level of TaPCS1 remained unaffected. The pretreatment of plants with MJ leads to a lower accumulation of cadmium in the roots and leaves of wheat. At the same time, MJ had no effect on the linear growth parameters of plants, but had a positive effect on the accumulation of biomass under cadmium. It was assumed that MJ is involved in plant tolerance to cadmium by increasing the expression of the TaGS1 and TaPCS1 genes and, as a result, enhancing the synthesis of chelating compounds, as well as by reducing the supply of metal ions to plants.

Silver Nanoparticles' Therapeutic Antibacterial, Antiproliferative, and Toxicological Effects.

Silver nanoparticles, among others, have broad-spectrum antimicrobial properties. Silver nanoparticles have suppressed dangerous microorganisms in medical and agricultural settings in several studies. Chemicals are harmful to humans and the environment, raising awareness of bioactive synthetic methods. These methods produce nanoparticles with better physicochemical qualities, stability, and toxicity. Biogenic nanoparticles can be made from bacterial and fungal byproducts that reduce and stabilize. Encapsulating these nanoparticles with biomolecules from the producing organisms may boost stability and biological activity. Nanoparticles' quick, clean, cheap, and ecological biologic manufacturing technique increases biocompatibility. Silver nanoparticles affect fish, algae, cell-based in vitro procedures, and microbes. Even though most of these studies were done quickly in well-regulated labs with much higher silver ion concentrations than in real life. Many silver types undergo long-term chemical transformation at extremely low levels (ng/L to g/L) in aquatic ecosystems. Thus, silver nanoparticles' environmental and health hazards need additional investigation. Recently detected antimicrobial silver at 10102 μg/mL. Multiple processes make silver nanoparticles dangerous. Basic (Ag0) and monovalent (Ag+) silver are most poisonous. Silver framework free ions affect silver toxicity. ROS damage DNA when elemental or zero-valent silver penetrates tissues. Packaged foods, contaminated water, swimming pools, antifouling, nasal and throat medicines, and other pharmaceuticals include silver nanoparticles. Consumption accumulates silver ions in subcutaneous fat. Prolonged exposure causes argyria-blue-gray skin. Silver inhibits Na+ and Cl absorption, disrupting electrolytes. Airborne silver nanoparticles may influence chronic pulmonary disease patients. Silver ions oxidize enzyme thiols, hindering electron transport and DNA replication. Ag+ rapidly damages DNA and RNA. Silver nanoparticle breakdown into silver ions creates germ-killing ROS. Silver nanoparticles are more hazardous than silver ions in the same atmosphere.

Kinetic Characterization of Cerium and Gallium Ions as Inhibitors of Cysteine Cathepsins L, K, and S.

Heavy metal ions can disrupt biological functions via multiple molecular mechanisms, including inhibition of enzymes. We investigate the interactions of human papain-like cysteine endopeptidases cathepsins L, K, and S with gallium and cerium ions, which are associated with medical applications. We compare these results with zinc and lead, which are known to inhibit thiol enzymes. We show that Ga3+, Ce3+, and Ce4+ ions inhibit all tested peptidases with inhibition constants in the low micromolar range (between 0.5 µM and 10 µM) which is comparable to Zn2+ ions, whereas inhibition constants of Pb2+ ions are one order of magnitude higher (30 µM to 150 µM). All tested ions are linear specific inhibitors of cathepsin L, but cathepsins K and S are inhibited by Ga3+, Ce3+, and Ce4+ ions via hyperbolic inhibition mechanisms. This indicates a mode of interaction different from that of Zn2+ and Pb2+ ions, which act as linear specific inhibitors of all peptidases. All ions also inhibit the degradation of insoluble elastin, which is a common target of these peptidases in various inflammatory diseases. Our results suggest that these ions and their compounds have the potential to be used as cysteine cathepsin inhibitors in vitro and possibly in vivo.

Chromatographic measurement of 3-hydroxyanthranilate 3,4-dioxygenase activity reveals that edaravone can mitigate the formation of quinolinic acid through a direct enzyme inhibition

Herein it is reported the development and application of two chromatographic assays for the measurement of the activity of 3-Hydroxyanthranilate-3,4-dioxygenase (3HAO). Such an enzyme converts 3-Hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic semialdehyde (ACMS), which undergo a spontaneous, non-enzymatic cyclization to produce quinolinic acid (QUIN). The enzyme activity was measured by quantitation of the substrate consumption over time either with spectrophotometric (UV) or mass spectrometric (MS) detection upon reversed-phase chromatographic separation. MS detection resulted more selective and sensitive, but less accurate and precise. However, both methods have sufficient sensitivity to allow the measurement of enzyme activity with consistent results compared to literature data. Since MS detection allowed less sample consumption it was used to calculate the kinetics parameters (i.e., Vmax and Kd) of recombinant 3HAO. Another MS-based method was then developed to measure the amount of QUIN produced, revealing an incomplete conversion of 3HAA to QUIN. As suggested by previous studies, the enzyme activity was apparently sensitive to the redox state of the enzyme thiols. In fact, thiol reducing agents such as dithiothreitol (DTT) and glutathione (GSH), can alter the enzyme activity although the investigation on the exact mechanism involved in such effect was beyond the scope of the research. Interestingly, edaravone (EDA) induced an in vitro suppression of QUIN production through direct, competitive 3HAO inhibition. EDA is a molecule approved for the treatment of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with an increase of QUIN concentrations in both serum and cerebrospinal fluid. Although EDA was reported to mitigate ALS progression its mode of action is still largely unknown. Some studies reported antioxidant and radical scavenger properties of EDA, but none confirm a direct activity as 3HAO enzyme inhibitor. Since QUIN is reported to be a neurotoxic metabolite, 3HAO inhibition can contribute to the beneficial effect of EDA in ALS, although such a mechanism must be then confirmed in vivo. However, EDA might be a convenient scaffold for the design of selective 3HAO inhibitors with potential applications in ALS treatment.

Evaluation of Oxidative Stress and Inflammatory Biomarkers Pre and Post-Treatment in New Diagnosed Atherosclerotic Patients

ABSTRACT Atherosclerosis is a chronic vascular inflammatory disease associated to oxidative stress and endothelial dysfunction. It is characterized by lipid accumulation in the arterial wall, increased hyperlipidemia, oxidative stress, lipid peroxidation, and protein oxidation. Our study included 45 patients ages of 40–60 and 45 healthy volunteers with similar demographic characteristics without any chronic disease as well. Fasting plasma glucose, BUN, creatinine, LDL-cholesterol, HDL-cholesterol, triglyceride, total cholesterol, HbA1c, and C-reactive protein (CRP) levels were measured using commercial kits by autoanalyzer. The oxidative stress biomarkers total oxidant status (TOS), total antioxidant status (TAS), total thiol (TT), native thiol (NT), catalase (CAT), paraoxonase (PON1), and arylesterase (ARES) enzyme activities were measured using photometric methods. The inflammatory biomarkers interleukin 1 beta (IL-1β), tumor necrosis factor-α (TNF-α), presepsin (PSPN), and raftlin (RFTN1) levels were measured with ELISA Kits. Oxidative stress index (OSI) and disulfide (DIS) were calculated. The clinical, biochemical biomarkers such as BUN, creatinine, HDL, LDL, total cholesterol, triglyceride, and CRP levels were found to be higher than the control group and lower post-treatment compared to the pre-treatment group (p <0.001). The oxidative stress parameters, TOS, OSI, and DIS levels were found to be higher than the control group, and the levels before the treatment were statistically significantly higher than after the treatment (p < 0.001).Antioxidant biomarkers TAS, TT, and NT levels were low in the patient group. Inflammatory biomarkers were highest before treatment and decreased with treatment. Oxidative stress and inflammation, which increased in atherosclerosis patients may guide disease prognosis and treatment strategies.

Evaluation of CBSX Proteins as Regulators of the Chloroplast Thioredoxin System.

The chloroplast-localized cystathionine β-synthase X (CBSX) proteins CBSX1 and CBSX2 have been proposed as modulators of thioredoxins (Trxs). In this study, the contribution of CBSX proteins to the redox regulation of thiol enzymes in the chloroplast Trx system was evaluated both in vitro and in vivo. The in vitro biochemical studies evaluated whether CBSX proteins alter the specificities of classical chloroplastic Trx f and Trx m for their target proteins. However, addition of CBSX proteins did not alter the specificities of Trx f and Trx m for disulfide bond reduction of the photosynthesis-related major thiol enzymes, FBPase, SBPase, and NADP-MDH. In vivo analysis showed that CBSX-deficient mutants grew similarly to wild type plants under continuous normal light conditions and that CBSX deficiency did not affect photo-reduction of photosynthesis-related thiol enzymes by Trx system at several light intensities. Although CBSX proteins have been suggested as modulators in the chloroplast Trx system, our results did not support this model, at least in the cases of FBPase, SBPase, and NADP-MDH in leaves. However, fresh weights of the cbsx2 mutants were decreased under short day. Since Trxs regulate many proteins participating in various metabolic reactions in the chloroplast, CBSX proteins may function to regulate other chloroplast Trx target proteins, or serve as modulators in non-photosynthetic plastids of flowers. As a next stage, further investigations are required to understand the modulation of Trx-dependent redox regulation by plastidal CBSX proteins.

Inside Back Cover: Cyclometalated Gold(III)‐Hydride Complexes Exhibit Visible Light‐Induced Thiol Reactivity and Act as Potent Photo‐Activated Anti‐Cancer Agents (Angew. Chem. Int. Ed. 27/2020)

A photoactivatable prodrug approach overcomes the deactivation of drugs based on gold complexes caused by off-target binding to serum albumin. In their Research Article on page 11046, T. Zou et al. show that gold(III) hydride complexes are inert towards serum albumin in the dark. Once they enter tumor tissues, their thiol reactivity can be photochemically activated, which leads to potent inhibition of tumor thiol enzymes through the formation of Au−S bonds.

Novel applications of modification of thiol enzymes and redox-regulated proteins using S-methyl methanethiosulfonate (MMTS)

S-Methyl methanethiosulfonate (MMTS) is used in experimental biochemistry for alkylating thiol groups of protein cysteines. Its applications include mainly trapping of natural thiol-disulfide states of redox-sensitive proteins and proteins which have undergone S-nitrosylation. The reagent can also be employed as an inhibitor of enzymatic activity, since nucleophilic cysteine thiolates are commonly present at active sites of various enzymes. The advantage of using MMTS for this purpose is the reversibility of the formation of methylthio mixed disulfides, compared to irreversible alkylation using conventional agents. Additional benefits include good accessibility of MMTS to buried protein cysteines due to its small size and the simplicity of the protection and deprotection procedures. In this study we report examples of MMTS application in experiments involving oxidoreductase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), redox-regulated protein (recoverin) and cysteine protease (triticain-α). We demonstrate that on the one hand MMTS can modify functional cysteines in the thiol enzyme GAPDH, thereby preventing thiol oxidation and reversibly inhibiting the enzyme, while on the other hand it can protect the redox-sensitive thiol group of recoverin from oxidation and such modification produces no impact on the activity of the protein. Furthermore, using the example of the papain-like enzyme triticain-α, we report a novel application of MMTS as a protector of the primary structure of active cysteine protease during long-term purification and refolding procedures. Based on the data, we propose new lines of MMTS employment in research, pharmaceuticals and biotechnology for reversible switching off of undesirable activity and antioxidant protection of proteins with functional thiol groups.

Evaluation of hematological, biochemical parameters and thiol enzyme activity in chrome plating workers.

The most commonly used solution in chrome plating bath is chromic acid (hexavalent Cr), and a considerable amount of mists is released into the air and consequently produce hazards to workers. Thus, the aim of this study was to evaluate whether the biomarker of exposure to metals, specially Cr levels, presents associations with hematological and biochemical parameters and if they can alter the activity of enzymes that contain thiol groups such as pyruvate kinase, creatine kinase, adenylate kinase, and δ-aminolevulinate dehydratase. Fifty male chrome plating workers were used for exposed group and 50 male non-exposed workers for control group. For that, biological monitoring was performed through quantification of metals on total blood and urine by inductively coupled plasma mass spectrometry (ICP-MS) and enzyme activity was performed by spectrometry in erythrocytes. In addition, chromium levels in water was quantified and ecotoxicology assay was performed with Allium cepa test. The results demonstrated that blood and urinary chromium levels in exposed group were higher than the control group (p < 0.0001). Furthermore, decreased activity of enzymes was found in those that contain thiol groups from exposed group when compared with the control group (p < 0.001). The water analysis did not present a statistical difference between control and exposed groups (p > 0.05), demonstrating that water did not seem to be the source of contamination. In summary, our findings indicated some toxicology effects observed in the exposed group, such as thiol enzyme inhibition, mainly associated with occupational exposure in chrome plating and besides the presence of other metals, and Cr demonstrated to influence the activity of the enzymes analyzed in this research.

Протеолитические ферменты и их белковые ингибиторы из зерна тритикале

Протеолитические ферменты играют значительную роль в процессах, протекающих при хранении и переработке растительного сырья. На современном этапе приоритетами любой пищевой технологии является сохранение нативных свойств биологически ценных компонентов сырья, обладающих лечебно-профилактическими свойствами, и модификация пищевого сырья с целью получения компонентов с определенными технологическими и функциональными свойствами. В настоящее время наибольшее распространение получили методы ферментативной модификации зернового сырья с использованием микробных ферментных препаратов. В то же время исследование собственных протеолитических ферментов, их выделение и очистка, с целью применения для модификации эндогенных белков, к которым они проявляют наибольшее сродство, представляется весьма интересной задачей, в плане использования всего биопотенциала зерна и “экологичности” получаемых продуктов. Такое исследование неразрывно сопряжено с изучением белковых ингибиторов протеиназ, которые представляют исключительный интерес как фактор регуляции процесса протеолиза. В статье приведены экспериментальные данные по выделению, частичной очистке и характеристике протеолитических ферментов зерна тритикале, действующих в кислой, нейтральной и щелочной областях рН. Показано, что кислые и нейтральные протеиназы зерна тритикале являются ферментами тиолового типа, Щелочные протеиназы относятся к сериновым протеиназам. Методом гель-хроматографии определена их молекулярная масса: 75000, 250000 и 50000 Да соответственно. Предложена схема выделения и очистки белковых ингибиторов эндогенных протеиназ из зерна тритикале. Показана их способность подавлять активность собственных протеиназ и трипсина. Проведено разделение ингибиторов трипсина и щелочных протеиназ тритикале (с молекулярной массой 30000 ÷ 35000 Да) и ингибиторов кислых и нейтральных протеиназ тритикале (с молекулярной массой 15000 ÷ 20000 Да). Частично очищенных препараты протеиназ и их белковых ингибиторов, полученные по предложенным схемам могут быть использованы для ферментативной модификации растительных белков и регуляции процессов протеолиза в различных пищевых технологиях, использующих зерновое сырье.

Protective effect of novel substituted nicotine hydrazide analogues against hypoxic brain injury in neonatal rats via inhibition of caspase

In hypoxic-ischemic injury of the brain of neonates, the level of caspase-3 was found to be aberrantly activated. Its overexpression leads to the alteration of cytoskeleton protein fodrin and loss of DNA repair enzyme which ultimately results in neurological impairment and disability. Concerning this, the present study was intended to develop novel nicotine hydrazide analogues as caspase inhibitors via efficient synthetic route. These compounds were subsequently tested for inhibitory activity against caspase-3 and -7 where they exhibit highly potent activity against caspase-3 revealing compound 5k as most potent inhibitor (IC50=19.4±2.5μM). In Western blot analysis, 5k considerably inhibits the overexpression of caspase-3. The aryl nicotinate of compound 5k, as indicated by molecular docking was found to engage His121 and critical enzyme thiols, i.e., Cys163 of caspase-3 for its potent activity. Moreover, histopathological examination of brain tissues and hippocampus neurons showed that compound 5k considerably improves the brain injury and exert neuroprotective effects in hypoxic-ischemic (HI). In brain homogenate, 5k significantly improves the activity of MDA, SOD, GSH-Px, CAT and T-AOC to exert its beneficial effect against oxidative stress induced by HI injury.

A convenient screening method to differentiate phenolic skin whitening tyrosinase inhibitors from leukoderma-inducing phenols

BackgroundTyrosinase is able to oxidize a great number of phenols and catechols to form ortho-quinones. Ortho-quinones are highly reactive compounds that exert cytotoxicity through binding with thiol enzymes and the production of reactive oxygen species. Certain phenolic (and catecholic) compounds are known to induce contact/occupational leukoderma through activation to ortho-quinones. ObjectiveWe report a convenient screening method to follow the oxidation of those leukoderma-inducing phenols by mushroom tyrosinase. MethodsOxidation of phenolic compounds by mushroom tyrosinase was followed periodically by UV–vis spectrophotometry. The production of ortho-quinones were confirmed by their absorptions around 400–420nm. HPLC analysis after reduction with NaBH4 detected the corresponding catechols. ResultsLeukoderma-inducing phenols, rhododendrol, raspberry ketone, 4-methoxyphenol, 4-benzyloxyphenol, 4-tert-butylphenol, and 4-tert-butylcatechol, were readily oxidized by mushroom tyrosinase to form ortho-quinones. On the other hand, phenolic skin whitening tyrosinase inhibitors, ellagic acid, 4-n-butylresorcinol, potassium 4-methoxysalicylate, and 2,2′-dihydroxy-5,5′-di-n-propylbiphenyl, were not oxidized by mushroom tyrosinase, while arbutin was only slowly oxidized. ConclusionThis study has provided a convenient screening method to differentiate phenolic skin whitening tyrosinase inhibitors from leukoderma-inducing phenols. A common chemical feature of the latter group of compounds is that they are readily oxidized by tyrosinase to form reactive ortho-quinone species. The present results point out the necessity that tyrosinase inhibitors should also be examined as substrates if they are phenolic compounds.

Interaction of adenanthin with glutathione and thiol enzymes: Selectivity for thioredoxin reductase and inhibition of peroxiredoxin recycling

The diterpenoid, adenanthin, represses tumor growth and prolongs survival in mouse promyelocytic leukemia models (Liu et al., Nat. Chem. Biol. 8, 486, 2012). It was proposed that this was done by inactivating peroxiredoxins (Prxs) 1 and 2 through the formation of an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidation to a disulfide-linked dimer. However, contrary to the original report, both the peroxidatic and the resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reacting with a second-order rate constant of 25±5M–1s–1. With 50µM adenanthin, the peroxidatic and resolving Cys of Prx2 reacted with half-times of 7 and 40min, respectively, compared with 10min for GSH. When erythrocytes or Jurkat T cells were treated with adenanthin, we saw no evidence for a reaction with Prxs 1 or 2. Instead, adenanthin caused time- and concentration-dependent loss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normally recycled by thioredoxin reductase and thioredoxin. Our results indicate that Prx reduction was inhibited. We observed rapid inhibition of purified thioredoxin reductase (half-time 5min with 2µM adenanthin) and in cells, thioredoxin reductase was much more sensitive than GSH and loss of both preceded accumulation of oxidized Prxs. Thus, adenanthin is not a specific Prx inhibitor, and its reported antitumor and anti-inflammatory effects are more likely to involve more general inhibition of thioredoxin and/or glutathione redox pathways.

Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana.

The thiol/disulfide redox network mediated by the thioredoxin (Trx) system in chloroplasts ensures light-responsive control of diverse crucial functions. Despite the suggested importance of this system, the working dynamics against changing light environments remains largely unknown. Thus, we directly assessed the in vivo redox behavior of chloroplast Trx-targeted thiol enzymes in Arabidopsis thaliana. In a time-course analysis throughout a day period that was artificially mimicked to natural light conditions, thiol enzymes showed a light-dependent shift in redox state, but the patterns were distinct among thiol enzymes. Notably, the ATP synthase CF(1-γ) subunit was rapidly reduced even under low-light conditions, whereas the stromal thiol enzymes fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and NADP-malate dehydrogenase were gradually reduced/re-oxidized along with the increase/decrease in light intensity. Photo-reduction of thiol enzymes was suppressed by the impairment of photosynthetic linear electron transport using DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but sensitivity to the impairment was uneven between CF(1-γ) and other stromal thiol enzymes. These different dependencies of photo-reduction on electron transport, rather than the redox state of Trx and the circadian clock, could readily explain the distinct diurnal redox behaviors of thiol enzymes. In addition, our results indicate that the cyclic electron transport around PSI is also involved in redox regulation of some thiol enzymes. Based on these findings, we propose an in vivo working model of the redox regulation system in chloroplasts.

Endocannabinoid Enzyme Engineering: Soluble Human Thio-Monoacylglycerol Lipase (sol-S-hMGL)

In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu(169);Leu(176))-to-Ser(Ser(169);Ser(176)) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser(122) with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser(122) residue as compared to that of Cys(122) in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys(122) residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser(122), and the mechanisms underlying hMGL inhibition by different classes of small molecules.

Luciferase inhibition by a novel naphthoquinone

The novel naphthoquinone 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) was created as a potential chemotherapeutic agent. Previous work showed it is an irreversible inhibitor of type I and II topoisomerases that alkylates specific enzyme thiols. While analyzing the effect of TU100 on cancer cells, we discovered it is a potent inhibitor of luciferase derived from both Photinus pyralis (fireflies) and Renilla reniformis (sea pansy). Pre-incubation experiments showed that TU100 does not irreversibly inactivate luciferase, indicating its mechanism is different from that observed with topoisomerases. Firefly luciferase generates light using ATP and luciferin as substrates (bioluminescence). An examination of TU100 inhibition at varying substrate concentrations revealed the drug is uncompetitive with respect to ATP and competitive with respect to luciferin. The TU100 binding constant ( K I ) is 2.5 ± 0.7 μM as determined by Dixon plot analysis. These data suggest TU100 specifically binds the luciferase–ATP complex and prevents its interaction with luciferin. Given the novel structure of TU100, unique mechanism of action, and ability to target luciferase from different species, these results identify TU100 as an important new reagent for investigating and regulating bioluminescent enzymes.

Enzymatic properties of the glycine d-alanine aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji)

The gdaA gene encoding S12 family glycine-D-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the D-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high D-stereospecificity and efficiently released N-terminal glycine and D-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and D-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and D-alanine aminopeptidase activity detected at a pH range of 6 to 9.

Catalytic site cysteines of thiol enzyme: sulfurtransferases.

Thiol enzymes have single- or double-catalytic site cysteine residues and are redox active. Oxidoreductases and isomerases contain double-catalytic site cysteine residues, which are oxidized to a disulfide via a sulfenyl intermediate and reduced to a thiol or a thiolate. The redox changes of these enzymes are involved in their catalytic processes. On the other hand, transferases, and also some phosphatases and hydrolases, have a single-catalytic site cysteine residue. The cysteines are redox active, but their sulfenyl forms, which are inactive, are not well explained biologically. In particular, oxidized forms of sulfurtransferases, such as mercaptopyruvate sulfurtransferase and thiosulfate sulfurtransferase, are not reduced by reduced glutathione but by reduced thioredoxin. This paper focuses on why the catalytic site cysteine of sulfurtransferase is redox active.

Hydrogen peroxide-induced inactivation of urease: Mechanism, kinetics and inhibitory potency

Abstract Evidence has been collected that H2O2 resulting from redox reactions taking place in some inhibitor systems, is responsible for the inactivation of urease. Accordingly, in this study the inactivation by H2O2 exogenously added to urease (jack bean) was investigated. The reaction accountable for the inactivation was the oxidation of the enzyme thiol groups. The reaction was studied at pHs over 6.2–8.2. At each pH, the first-order kinetics was obeyed, the resulting dependences of kobs on H2O2 concentration being hyperbolic. Analyzed with Kitz-Wilson method, the kinact appeared to be invariant with pH, while the inhibitor binding constant KI decreased with increasing pH. Consequently, the process grew faster with an increase in pH, the second-order rate constants increasing from 0.0032 M−1 s−1 at pH 6.2 to 0.0615 M−1 s−1 at pH 8.2, consistent with higher susceptibility of thiolate anions to oxidation by H2O2 compared to reduced thiols. The reactions always resulted in irreversible inactivation of urease, indicative of the oxidation of thiol groups to either sulfinic or sulfonic acid, independent of the extent of inactivation. Analysis of the numbers of urease thiol groups modified with H2O2 revealed that for the complete inactivation, out of 36 thiols/molecule available under non-denaturating conditions, the rapid oxidation of 30 highly reactive –SH groups was responsible for a 50% loss in enzyme activity, and the slower oxidation of the remaining six –SH groups, for the other 50%. The enzyme was protected by active-site binding inhibitors, boric acid and fluoride, from the inactivation, suggesting that the six thiols are the active-site flap Cys-592s. For comparison with other urease inhibitors, the IC50 was determined for 20 min incubation with H2O2, its value changing from 242 mM at pH 6.2 to 11 mM at pH 8.2.

Disulfiram is a DNA demethylating agent and inhibits prostate cancer cell growth

The clinical success of the nucleoside analogs 5-aza-cytidine (5-azaC) and 5-aza-2'deoxycytidine (5-aza-dC) as DNA methyltransferase (DNMT) inhibitors has spurred interest in the development of non-nucleoside inhibitors with improved pharmacologic and safety profiles. Because DNMT catalysis features attack of cytosine bases by an enzyme thiol group, we tested whether disulfiram (DSF), a thiol-reactive compound with known clinical safety, demonstrated DNMT inhibitory activity. Inhibition of DNMT1 activity by DSF was assessed using methyltransferase activity assays with recombinant DNMT1. Next, prostate cancer cell lines were exposed to DSF and assessed for: i) reduction of global 5-methyl cytosine ((5me)C) content using liquid chromatography/tandem mass spectrometry (LC-MS/MS); ii) gene-specific promoter demethylation by methylation-specific PCR (MSP); and iii) gene-reactivation by real-time RT-PCR. DSF was also tested for growth inhibition using prostate cancer cell lines propagated in vitro in cell culture and in vivo as xenografts in nude mice. Disulfiram showed a dose-dependent inhibition of DNMT1 activity on a hemimethylated DNA substrate. In prostate cancer cells in culture, DSF exposure led to reduction of global genomic (5me)C content, increase in unmethylated APC and RARB gene promoters, and associated re-expression of these genes, but did not significantly alter prostate-specific antigen (PSA) expression. DSF significantly inhibited growth and clonogenic survival of prostate cancer cell lines in culture and showed a trend for reduced growth of prostate cancer xenografts. Disulfiram is a non-nucleoside DNMT1 inhibitor that can reduce global (5me)C content, reactivate epigenetically silenced genes, and significantly inhibit growth in prostate cancer cell lines.