Irreversible Inactivation Research Articles

Acetylene containing cyclo(L-Tyr-L-Tyr)-analogs as mechanism-based inhibitors of CYP121A1 from Mycobacterium tuberculosis

Tuberculosis (TB) is a globally significant infective disease that is caused by a single infectious agent, Mycobacterium tuberculosis (Mtb). Because of the rise in the number of multidrug-resistant (MDR) TB strains, identification of alternative drug targets for the development of drugs with different mechanism of actions is desired. CYP121A1, one of the twenty cytochrome P450 enzymes encoded in the Mtb genome, was previously shown to be essential for bacterial growth. This enzyme catalyzes the intramolecular C–C crosslinking reaction of the cyclopeptide cyclo(L-tyr-L-tyr) (cYY) yielding the metabolite mycocyclosin. In the present study, acetylene-substituted cYY-analogs were synthesized and evaluated as potential mechanism-based inhibitors of CYP121A1. The acetylene-substituted cYY-analogs were capable of binding to CYP121A1 with affinities comparable with cYY, and exhibited a Type I binding mode, indicative of a substrate-like binding, mandatory for metabolism. Only the cYY-analogs which contain an acetylene-substitution at one (2a) or both (3) para-positions of cYY showed mechanism-based inhibition of CYP121A1 activity. The values of KI and kinact were 236 µM and 0.045 min−1, respectively, for compound 2a, and 145 µM and 0.015 min−1, repectively, for compound 3 The inactivation could neither be reversed by dialysis nor be prevented by including glutathione. LC-MS analysis demonstrated that the inactivation results from covalent binding to the apoprotein, whereas the heme was unmodified. Interestingly, the mass increment of the CYP121A1 apoprotein was significantly smaller than was expected from the ketene formed by oxidation of the acetylene-group, indicative for a secondary cleavage reaction in the active site of CYP121A1. Although the two acetylene-containing cYY-analogs showed significant mechanism-based inhibition, growth inhibition of the Mtb strains was only observed at millimolar concentrations. This low efficacy may be due to insufficient irreversible inactivation of CYP121A1 and/or insufficient cellular uptake. Although the identified mechanism-based inhibitors have no perspective for Mtb-treatment, this study is the first proof-of-principle that mechanism-based inhibition of CYP121A1 is feasible and may provide the basis for new strategies in the design and development of compounds against this promising therapeutic target.

Kinetics and thermodynamics of catalysis and thermal inactivation of a novel α-amylase (Tp-AmyS) from Thermotoga petrophila

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned in pHIS-parallel1 expression vector and overexpressed in Escherichia coli. The steady-state kinetic parameters (Vmax, Km, kcat and kcat/Km) for the hydrolysis of amylose (1.39 mg/min, 0.57 mg, 148.6 s−1, 260.7), amylopectin (2.3 mg/min, 1.09 mg, 247.1 s−1, 226.7), soluble starch (2.67 mg/min, 2.98 mg, 284.2 s−1, 95.4) and raw starch (2.1 mg/min, 3.6 mg, 224.7 s−1, 61.9) were determined. The activation energy (Ea), free energy (ΔG), enthalpy (ΔH) and entropy of activation (ΔS) at 98 °C were 42.9 kJ mol−1, 74 kJ mol−1, 39.9 kJ mol−1 and −92.3 J mol−1 K−1, respectively, for soluble starch hydrolysis. While ΔG of substrate binding (ΔGE-S) and ΔG of transition state binding (ΔGE-T) were 3.38 and −14.1 kJ mol−1, respectively. Whereas, EaD, Gibbs free energy (ΔG*), increase in the enthalpy (ΔH*) and activation entropy (ΔS*) for activation of the unfolding of transition state were 108, 107, 105 kJ mol−1 and −4.1 J mol−1 K−1. The thermodynamics of irreversible thermal inactivation of Tp-AmyS revealed that at high temperature the process involves the aggregation of the protein.

Kinetic and thermodynamic parameters of nisin thermoinactivation

Understanding the thermal inactivation of biopreservatives in food matrixes is essential for their proper use in the food industry, enabling reduction of heating time and optimization of heating temperature. With this aim in mind, the effect of heat treatment on the activity and thermostability of commercial nisin from Lactococcus lactis was investigated. Residual activity tests performed for 90 min in the 70–86 °C temperature range at pH 3.0 demonstrated that bacteriocin thermoinactivation followed first-order kinetics. Low values of the inactivation rate constant (0.0089–0.0892 min−1) and high values of the decimal reduction time (25.8 ≤ D-value ≤ 258.4 min) indicated excellent nisin thermostability in the lower end of the investigated temperature range, but the thermal resistance constant (z-value) was only 15.4 °C, which pointed out poor thermoresistance at the higher end. Thermodynamic parameters estimated for nisin irreversible thermal inactivation were energy, enthalpy and Gibbs free energy of activation of 155.1, 152.2–152.3 and 83.5–86.6 kJ mol−1, respectively, and activation entropy of 188.6–192.5 J mol−1 K−1. If on the one hand these results taken together confirm the thermostability of nisin as a biopreservative in use in the food industry, on the other hand suggest the importance of looking for new more thermostable antimicrobials to use in operations at higher temperatures.

Bioluminescence Profiling of NanoKAZ/NanoLuc Luciferase Using a Chemical Library of Coelenterazine Analogues.

We describe here an extensive structure-bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 O-acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provided a 1.5-fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal-to-background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate-dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer-dependent fraction of a photon).

Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors.

Dual-acting virucidal entry inhibitors (DAVEIs) have previously been shown to cause irreversible inactivation of HIV-1 Env-presenting pseudovirus by lytic membrane transformation. This study examined whether this transformation could be generalized to include membranes of Env-presenting cells. Flow cytometry was used to analyze HEK293T cells transiently transfected with increasing amounts of DNA encoding JRFL Env, loaded with calcein dye, and treated with serial dilutions of microvirin (Q831K/M83R)-DAVEI. Comparing calcein retention against intact Env expression (via Ab 35O22) on individual cells revealed effects proportional to Env expression. "Low-Env" cells experienced transient poration and calcein leakage, while "high-Env" cells were killed. The cell-killing effect was confirmed with an independent mitochondrial activity-based cell viability assay, showing dose-dependent cytotoxicity in response to DAVEI treatment. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and cytotoxicity, further reinforcing the Env dependence of the observed effect. Controls with unlinked DAVEI components showed no effect on calcein leakage or cell viability, confirming a requirement for covalently linked DAVEI compounds to achieve Env transformation. These data demonstrate that the metastability of Env is an intrinsic property of the transmembrane protein complex and can be perturbed to cause membrane disruption in both virus and cell contexts.

Are Aspirin and Apixaban Sufficient to Prevent Immunothrombosis in COVID-19?

Severe COVID-19 disease is associated with respiratory and vascular injury and microvascular immunothrombosis mediated by complement activation, inflammatory lipid storm, platelet activation, platelet-leukocyte adhesion and activation of coagulation cascade. Amongst the inflammatory lipids, thromboxane A2 (TxA2) is a known key mediator of microvascular thrombosis. The levels of TxB2, a stable metabolite of TxA2 are markedly increased in the bronchoalveolar lavage fluid and plasma in severe COVID-19 patients. Low-dose aspirin mitigates the generation of prostanoids including TxA2 by irreversible inactivation of the constitutive enzyme cyclooxygenase (COX)-1. The anti-thrombotic action of aspirin is currently being investigated in outpatient and hospitalized COVID-19 patients in the global RECOVERY and ACTIV-4 clinical trials. Several lines of investigations suggest that COX-2 plays an important and critical role in the immunothrombotic effects mediated by COVID-19. Pharmacologic inhibition of either COX-1 or COX-2 can prevent a plethora of lipid mediators of inflammation that are both pro- and anti-inflammatory in function. Thus, a more definitive approach to prevent immunothrombotic events in COVID-19 will be to directly block the prothrombotic effects of TxA2. Although thromboxane synthase (TS) inhibitors suppress TxA2 formation, accumulation of the substrate PGH2 is known to interact with the platelet and vessel wall TxA2 prostanoid receptor (TPR), thus reducing the antiplatelet effects of TS inhibitors. TPR antagonists block the activity of both TxA2 and PGH2 on platelets and vessels but do not block TxA2 production, which leads to increased generation of 11-dehydro-thromboxane B2, a stable metabolite of TxA2, and a potent agonist of the DP2 (CRTH2) receptor for prostaglandin D2 (PGD2). PGD2/DP2 receptor signaling has been implicated in immune dysregulation in viral infections including COVID-19. Ramatroban, an orally bioavailable, potent, dual TxA2/TP and PGD2/DP2 receptor antagonist, has demonstrated efficacy in a variety of animal models of atherosclerosis, thrombosis and sepsis. Ramatroban has a proven safety profile, having been used in Japan over the past 20 years as a treatment of allergic rhinitis and therefore, merits investigation as a promising antithrombotic and immunomodulator agent for chemoprophylaxis and treatment in COVID-19 patients.

Effects of application of ultrasound waves in the shelf life of an amazon fruit: araçá-boi

The aim of this work was to evaluate the effects of ultrasonic treatments in quality of araçá-boi fruits ( Eugenia stipitata McVaugh) during storage. Araçá-boi fruits were processed at a constant ultrasonic frequency of 40 kHz at 5 and 15 min. In the sequence, they were divided in three groups: control (CG), 5 min (G5M) and 15 min (G15M) of ultrasonic treatment and stored at room temperature for six days. The quality of the untreated and treated fruits was evaluated at 0, 3 and 6 days of storage and the following quality parameters were analyzed: weight loss, vitamin C, pH, titratable acidity, soluble solid content, total and reducing sugars. The maximum shelf life of fruits was between 3 and 6 days after harvest, for the three groups. The exposure time to the ultrasonic treatment influenced the quality of the fruits. The fruits of the group G5M presented less degradation, for some parameters (weight loss, pH, titratable acidity and concentration of sugars). In contrast, the fruit of the group G15M showed accelerated degradation. The effect of the cavitation phenomenon provoked by ultrasound treatment cause the irreversible destruction and inactivation of enzymes. Thus, it is assumed that 5 min of the treatment may have been more effective in reducing the action of some enzymes, and with15 min, the cavitation phenomenon may have been excessive for cell tissue and caused degradation and anticipated senescence. The results of this study suggest that ultrasonic treatment can be effective in preserving the quality of fruits, but, the treatment time needs to be better investigated.

Inhibition of multidrug resistance related protein by acetyl salicylic acid in cultured human lung cells

BACKGROUND: Multidrug resistance related proteins (MRP-proteins), members of the ATP binding cassette (ABC-) superfamily, are known to play an important role in regulation of glutathione homeostasis within cells, and hence in GSH dependent transport processes of xenobiotics. Many airborne compounds are hazardous inducing inflammatory reactions in lung and are suspected to play a substantial role in initiation and/or promotion of tumor formation. It is interesting to clarify whether and how anti-prostaglandins modify MRP function in normal human lung cells. Aspirin, an acetylated salicylate, is classified among the non-steroidal anti-inflammatory drugs (NSAIDs), reduce inflammatory signs and symptoms exhibiting a broad range of pharmacological activities (analgesic, antipyretic and antiplatelet properties). Cyclooxygenase (COX)-system catalyses the rate limiting step in prostanoids (prostaglandin (PG)) synthesis and is also involved in drug resistance and poor prognosis of many neoplastic diseases. Aspirin's ability to suppress prostaglandins and thromboxanes production is due to its irreversible inactivation of the cyclo-oxygenase (COX) enzyme. Furthermore, transport activity study of MRP1 in NHBEC, PLC and A549 under the effect of exogenously supplied PGE2 showed an increased activity. METHODOLOGY and RESULTS: In this study, both the localisation and description of the cellular distribution of MRP-1 under standard culture conditions in normal human bronchial epithelial cells (NHBEC), peripheral lung cells (PLC) and tumor lung cells (A549) was proved using indirect immunofluorescence microscopy. All substrates and incubation periods have been pre-checked for cellular toxicity in the MTT assay in order to guarantee that non toxic conditions are applied. CONCLUSION: COX inhibitor (acetylsalicylic acid 1,2mM) could significantly decrease the transport activity of MRP1 in NHBEC, PLC and A549 in 1 and 3 days trials.

Development of immobilized laccase-based time temperature indicator by electrospinning zein fiber

Abstract To increase the stability of enzymatic time–temperature indicator (TTI), laccase was immobilized on the electrospun zein fiber (ceZL) with high specific surface area (11.579 m2/g). The ceZL exhibited a high relative activity within 4 °C – 40 °C and the activation energy (Ea) of coloration approached 26.28 kJ/moL for 80 μg/cm2 of ceZL-immobilized laccase. In dynamic temperature response experiments of ceZL using intermittent temperature changes between 4 °C and 25 °C, irreversible inactivation did not occur, indicating the desirable tolerance and sensitivity of ceZL to temperature alterations. Approximately 6 or 26 days was required to reach the endpoint of coloration at 4 °C when 25 or 8 μg/cm2 of laccase was immobilized on ceZL, respectively. The regression equation derived from kinetic analysis was conducted and could be further utilized to design a suitable TTI to monitor food quality according to the color change of TTI by using ceZL.

Reactions of Microorganisms with Atomic Oxygen Radical Anions: Damage of Cells and Irreversible Inactivation

Reactive oxygen species play important effects on organisms not onlyin vivobut alsoin vitro. The atomic oxygen radical anion (O-) has shown extremely high oxidation and reactivity towards small molecules of hydrocarbons. However, the O-effects on cells of microorganisms are scarcely investigated. This work showed the evidence that O-could react quickly with microorganisms (Escherichia coli,Bacillus subtilis,Staphylococcus aureus,Aspergillus niger,Saccharomyces cerevisiae, andActinomycetes(5046)) and damaged the cell walls seriously as well as their intrinsic structures, arising a fast and irreversible inactivation. SEM and TEM micrographs were used to reveal the structure changes of cells before and after reacting with O-radicals. The inactivation efficiencies of the microorganisms depended on the O-intensity, the initial population of microorganisms, the exposed area, the environment, and the microorganisms’ types. Over 99% reduction of an initial1.0×107colony-forming unit (cfu),E. colipopulation only required less than 2 minutes while exposed to a 0.23 μA/cm2O-flux under dry argon atmosphere (30°C, 1 atm). The observation of anionic intermediates (CO-, CO2-, H2O-, and anionic hydrocarbons) by time-of-flight (TOF) mass spectrometry and the neutral volatile products (CO, CO2, and H2O) by quadrupole mass spectrometry (Q-MS) provided an evidence of the reactions of O-with hydrocarbon bonds of the microorganisms. The inactivation mechanism of microorganisms induced by O-was discussed.

Numerical Analysis of Time-Dependent Inhibition by MDMA.

Methylenedioxymethamphetamine (MDMA) is a known drug of abuse and schedule 1 narcotic under the Controlled Substances Act. Previous pharmacokinetic work on MDMA used classic linearization techniques to conclude irreversible mechanism-based inhibition of CYP2D6. The current work challenges this outcome by assessing the possibility of two alternative reversible kinetic inhibition mechanisms known as the quasi-irreversible (QI) model and equilibrium model (EM). In addition, progress curve experiments were used to investigate the residual metabolism of MDMA by liver microsomes and CYP2D6 baculosomes over incubation periods up to 30 minutes. These experiments revealed activity in a terminal linear phase at the fractional rates with respect to initial turnover of 0.0354 ± 0.0089 in human liver microsomes and 0.0114 ± 0.0025 in baculosomes. Numerical model fits to percentage of remaining activity (PRA) data were consistent with progress curve modeling results, wherein an irreversible inhibition pathway was found unnecessary for good fit scoring. Both QI and EM kinetic mechanisms fit the PRA data well, although in CYP2D6 baculosomes the inclusion of an irreversible inactivation pathway did not allow for convergence to a reasonable fit. The kinetic complexity accessible to numerical modeling has been used to determine that MDMA is not an irreversible inactivator of CYP2D6, and instead follows what can be generally referred to as slowly reversible inhibition. SIGNIFICANCE STATEMENT: The work herein describes the usage of computational models to delineate between irreversible and slowly reversible time-dependent inhibition. Such models are used in the paper to analyze MDMA and classify it as a reversible time-dependent inhibitor.

Loss of Specific Active-Site Iron Atoms in Oxygen-Exposed [FeFe]-Hydrogenase Determined by Detailed X-ray Structure Analyses.

The [FeFe]-hydrogenases catalyze the uptake and evolution of hydrogen with unmatched speed at low overpotential. However, oxygen induces the degradation of the unique [6Fe-6S] cofactor within the active site, termed the H-cluster. We used X-ray structural analyses to determine possible modes of irreversible oxygen-driven inactivation. To this end, we exposed crystals of the [FeFe]-hydrogenase CpI from Clostridium pasteurianum to oxygen and quantitatively investigated the effects on the H-cluster structure over several time points using multiple data sets, while correlating it to decreases in enzyme activity. Our results reveal the loss of specific Fe atoms from both the diiron (2FeH) and the [4Fe-4S] subcluster (4FeH) of the H-cluster. Within the 2FeH, the Fe atom more distal to the 4FeH is strikingly more affected than the more proximal Fe atom. The 4FeH interconverts to a [2Fe-2S] cluster in parts of the population of active CpIADT, but not in crystals of the inactive apoCpI initially lacking the 2FeH. We thus propose two parallel processes: dissociation of the distal Fe atom and 4FeH interconversion. Both pathways appear to play major roles in the oxidative damage of [FeFe]-hydrogenases under electron-donor deprived conditions probed by our experimental setup.

The liquid fraction from hydrothermal pretreatment of wheat straw provides lytic polysaccharide monooxygenases with both electrons and H2O2 co-substrate

BackgroundEnzyme-aided valorization of lignocellulose represents a green and sustainable alternative to the traditional chemical industry. The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important components of the state-of-the art enzyme cocktails for cellulose conversion. Yet, these monocopper enzymes are poorly characterized in terms of their kinetics, as exemplified by the growing evidence for that H2O2 may be a more efficient co-substrate for LPMOs than O2. LPMOs need external electron donors and one key question of relevance for bioprocess development is whether the required reducing power may be provided by the lignocellulosic substrate.ResultsHere, we show that the liquid fraction (LF) resulting from hydrothermal pretreatment of wheat straw supports LPMO activity on both chitin and cellulose. The initial, transient activity burst of the LPMO reaction was caused by the H2O2 present in the LF before addition of LPMO, while the steady-state rate of LPMO reaction was limited by the LPMO-independent production of H2O2 in the LF. H2O2 is an intermediate of LF oxidation as evidenced by a slow H2O2 accumulation in LF, despite high H2O2 production rates. This H2O2 scavenging ability of LF is important since high concentrations of H2O2 may lead to irreversible inactivation of LPMOs.ConclusionsOur results support the growing understanding that fine-tuned control over the rates of H2O2 production and consumption in different, enzymatic and non-enzymatic reactions is essential for harnessing the full catalytic potential of LPMOs in lignocellulose valorization.

Association with lipids or detergents is essential for preservation of the active structure of lipoprotein-associated phospholipase A2

Recombinant lipoprotein-associated phospholipase A2 (rLp-PLA2) expressed in HEK293 cells has a propensity to form oligomers in the absence of detergents. Dilution of rLp-PLA2 in the absence of detergents results in irreversible inactivation of the enzyme. The monomeric rLp-PLA2 may expose its hydrophobic interfacial binding region or substrate binding compartment to water and that may cause structural collapsing of the enzyme. Formation of self-aggregate, complex with binding partners or association with detergent micelles is to block the access of aqueous solvent to the hydrophobic substrate binding site and therefore prevents the structural collapsing. Dilution inactivation of the enzyme can be prevented in the presence of LDL or HDL suggesting that Lp-PLA2 association with lipoprotein particles (LDL and HDL) is necessary for Lp-PLA2 to maintain its enzymatic activity in human plasma. Formation of higher affinity complex gave better protection of rLp-PLA2 structure and activity. The method can be harnessed to detect the interaction between rLp-PLA2 and components of lipoprotein particles. Apo(a), ApoB 100 and ApoA1 were found to protect the enzyme from inactivation at roughly the similar level (˜80 ± 5%) comparing to human serum albumin control (˜40%). One mg/ml pig brain phospholipid showed 100% protection under the same conditions.

A conserved motif liganding the [4Fe–4S] cluster in [4Fe–4S] fumarases prevents irreversible inactivation of the enzyme during hydrogen peroxide stress

Organisms have evolved two different classes of the ubiquitous enzyme fumarase: the [4Fe–4S] cluster-containing class I enzymes are oxidant-sensitive, whereas the class II enzymes are iron-free and therefore oxidant-resistant. When hydrogen peroxide (H2O2) attacks the most-studied [4Fe–4S] fumarases, only the cluster is damaged, and thus the cell can rapidly repair the enzyme. However, this study shows that when elevated levels of H2O2 oxidized the class I fumarase of the obligate anaerobe Bacteroides thetaiotaomicron (Bt-Fum), a hydroxyl-like radical species was produced that caused irreversible covalent damage to the polypeptide. Unlike the fumarase of oxygen-tolerant bacteria, Bt-Fum lacks a key cysteine residue in the typical “CXnCX2C″ motif that ligands [4Fe–4S] clusters. Consequently H2O2 can access and oxidize an iron atom other than the catalytic one in its cluster. Phylogenetic analysis showed that certain clades of bacteria may have evolved the full “CXnCX2C″ motif to shield the [4Fe–4S] cluster of fumarase. This effect was reproduced by the construction of a chimeric enzyme. These data demonstrate the irreversible oxidation of Fe–S cluster enzymes and may recapitulate evolutionary steps that occurred when microorganisms originally confronted oxidizing environments. It is also suggested that, if H2O2 is generated within the colon as a consequence of inflammation or the action of lactic acid bacteria, the inactivation of fumarase could potentially impair the central fermentation pathway of Bacteroides species and contribute to gut dysbiosis.

Benextramine and derivatives as novel human monoamine oxidases inhibitors: an integrated approach.

The two human monoamine oxidase isoforms (namely MAO A and MAO B) are enzymes involved in the catabolism of monoamines, including neurotransmitters, and for this reason are well-known and attractive pharmacological targets in neuropsychiatric and neurodegenerative diseases, for which novel pharmacological approaches are necessary. Benextramine is a tetraamine disulfide mainly known as irreversible α-adrenergic antagonist, but able to hit additional targets involved in neurodegeneration. As the molecular structures of monoamine oxidases contain nine cysteine residues, the aim of this study was to evaluate benextramine and eleven structurally related polyamine disulfides as potential MAO inhibitors. Most of the compounds were found to induce irreversible inactivation of MAOs with inactivation potency depending on both the polyamine structure and the enzyme isoform. The more effective compounds generally showed preference for MAO B. Structure-activity relationships studies revealed the key role played by the disulfide core of these molecules in the inactivation mechanism. Docking experiments pointed to Cys323, in MAO A, and Cys172, in MAO B, as target of this type of inhibitors thus suggesting that their covalent binding inside the MAO active site sterically impedes the entrance of substrate towards the FAD cofactor. The effectiveness of benextramine in inactivating MAOs was demonstrated in SH-SY5Y neuroblastoma cell line. These results demonstrated for the first time that benextramine and its derivatives can inactivate human MAOs exploiting a mechanism different from that of the classical MAO inhibitors and could be a starting point for the development of pharmacological tools in neurodegenerative diseases.

Biochemical characterization and kinetic/thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase with transfructosylating activity.

This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45-65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol-1 , and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3-290.4 kJ mol-1 , 568.7-571.0 J mol-1 K-1 , and 97.9-108.8 kJ mol-1 , respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.

Carbonic anhydrase CAH3 supports the activity of photosystem II under increased pH

The lumenal carbonic anhydrase (CA) CAH3 from green alga Chlamydomonas reinhardtii is the only one CA identified so far in close association with the photosystem II (PSII) multi-subunit protein complex. It was proposed earlier, that CAH3 could facilitate the H+ removal from the active center of the PSII water-oxidizing complex (WOC) under the light, thereby increasing its activity. In the present work, using PSII enriched membranes from the wild type of C. reinhardtii and from the CAH3-deficient mutant cia3, we demonstrate, that the suppression of the photosynthetic activity of PSII by increased pH is more pronounced in preparations from cia3 as compared to the wild type. Experiments with CA inhibitors show that the activity of CAH3 supports the function of PSII and prevents its irreversible inactivation under light upon increased pH. The photosynthetic activity of PSII from cia3 can be restored to the wild type level upon increased pH if an excess of HCO3– is added. These findings testify that the main role of CAH3 in the vicinity of PSII is the acceleration of the HCO3– dehydration reaction. Measurements of the photoinduced electron transfer rate in PSII from water or from an artificial electron donor indicate, that CAH3 has a direct influence on the WOC function. Based on the data obtained in this work we conclude, that in vivo CA-activity of CAH3 may support the photosynthetic activity of PSII at increased pH in the thylakoid lumen and can be observed under the dark to light transition.

The nature of β-cyclodextrin inhibition of potato polyphenol oxidase-catalyzed reactions

There is general interest in strategies to control polyphenol oxidase (PPO)-initiated enzymatic browning because it is often associated with declining food quality. Cyclodextrins are cyclic glucan oligosaccharides that form inclusion complexes with a number of PPO substrates. This study focuses on the effect of β-cyclodextrins (βCyD) on PPO-catalyzed reactions. Potato enzyme extracts and semi-purified potato PPO served as enzyme sources. Substrates included phenolics endogenous to potatoes. Reaction time-courses were followed spectrophotometrically; rates were compared by analysis of variance. Extents of βCyD inhibition of PPO-catalyzed reactions are shown to be substrate specific and can be quantitatively accounted for based on degrees of βCyD substrate sequestration. There was no evidence for direct irreversible βCyD inactivation of potato PPO. An apparent “direct PPO inactivation” by βCyD is shown to result from a sequence of sequestration-dependent reactions that occur in commonly employed assay systems for the quantification of PPO in fruits and vegetables.

A Receptor Model With Binding Affinity, Activation Efficacy, and Signal Amplification Parameters for Complex Fractional Response Versus Occupancy Data.

In quantitative pharmacology, multi-parameter receptor models are needed to account for the complex nonlinear relationship between fractional occupancy and response that can occur due to the intermixing of the effects of partial receptor activation and post-receptor signal amplification. Here, a general two-state receptor model and corresponding quantitative forms are proposed that unify three distinct processes, each characterized with its own parameter: 1) receptor binding, characterized by K d, the equilibrium dissociation constant used for binding affinity; 2) receptor activation, characterized by an (intrinsic) efficacy parameter ε; and 3) post-activation signal transduction (amplification), characterized by a gain parameter γ. Constitutive activity is accommodated via an additional ε R0 parameter quantifying the activation of the ligand-free receptor. Receptors can be active or inactive in both their ligand-free and ligand-bound states (two-state receptor theory), but ligand binding alters the likelihood of activation (induced fit). Because structural data now confirm that for most receptors in their active conformation, the small-molecule ligand-binding site is buried inside, straightforward binding to the active form (direct conformational selection) is unlikely. The proposed general equation has parameters that are more intuitive and better suited for optimization by nonlinear regression than those of the operational (Black and Leff) or del Castillo–Katz model. The model provides a unified framework for fitting complex data including a) fractional responses that do not match independently measured fractional occupancies, b) responses measured after partial irreversible inactivation of the “receptor reserve” (Furchgott method), c) fractional responses that are different along distinct downstream pathways (biased agonism), and d) responses with constitutive receptor activity. Furthermore, unlike previous models, the present one can be reduced back for special cases of its parameters to consecutively nested simplified forms that can be used on their own when adequate (e.g., ε R0 = 0, no constitutive activity; γ = 1: E max model for partial agonism; ε = 1: Clark equation).