Mechanism Of Enzyme Inactivation Research Articles

One-Step Suicide Substrate Inactivation Kinetics of a Ping-Pong Reaction with One Substrate Undergoing Disproportionation: A Theoretical Approach with Approximate Solutions

Understanding the kinetic mechanism of enzyme inactivation by suicide substrate is of relevance for the optimal design of new drugs with pharmacological and therapeutic applications. Suicide substrate inactivation usually occurs via a two-step mechanism, although there are enzymes such as peroxidase and catalase in which the suicide inactivation by H2O2 happens in a single step. The approximate solution of the ordinary differential equation (ODE) system of the one step suicide substrate inactivation kinetics for a uni–uni reaction following the irreversible Michaelis–Menten model was previously analytically solved when accumulation of the substrate–enzyme complex was negligible, however not for more complex models, such as a ping-pong reaction, in which the enzyme is present in two active states during the catalytic turnover. To solve this issue, a theoretical approach was followed, in which the standard quasi-steady state and reactant stationary approximations were invoked. These approximations allowed for solving the ODE system of a ping-pong reaction with one substrate undergoing disproportionation when suicide inactivation was also present. Although the approximate analytical solutions were rather unwieldy, they were still valuable in qualitative analyses to explore the time course of the reaction products and identify the enzyme active state that irreversibly reacted with the suicide substrate during the reaction.

Effect of pH on pulsed light inactivation of polyphenol oxidase

The inactivation of diverse food enzymes by pulsed light (PL) has been described before, including the inactivation of polyphenol oxidase (PPO) (at pH 6.5). Since the pH affects the conformation of enzymes, it may influence the inactivation of enzymes by PL. The aim of this work was to evaluate the effect of pH on the kinetics of the PL-inactivation and associated structural changes of a case enzyme. To this, PPO was treated by PL at different pHs (4.0–6.5) and its inactivation kinetics and changes in its structure were evaluated by spectrophotometric and spectrofluorometric methods. The inactivation proceeded faster at low pH and was highly correlated with the decrease in peak intrinsic fluorescence intensity. Phase diagrams and parameter A evolution indicated the absence of intermediate unfolded states during the course of the inactivation. No protein aggregation was detected by turbidimetry. Results indicate that although a low pH favors the PL-inactivation of PPO, the mechanism of inactivation is pH-independent. Beyond the specific outcome for PPO, the results are evidence of a general pH-independence in the mechanism of enzyme inactivation by PL in the pH range 4.0–6.5 and acidification can be a strategy to decrease treatment times during PL processing.

Changing the residues interaction pattern as a universal mechanism for enzyme inactivation and denaturation in supercritical CO2

Enzyme-catalyzed reactions in supercritical CO2 conditions bear many biotechnological advantages. However, many enzymes cannot function in this dense fluid due to the possible inactivation and denaturation. The molecular mechanism of enzyme inactivation in supercritical CO2 has been very controversial due to the limitations of experiments to examine macromolecule structure at the molecular levels in supercritical conditions. Most of hypotheses insist on the unique role of lysine residues in enzyme denaturation in supercritical CO2. Here, using molecular dynamics simulations and lysozyme as a model enzyme, we challenged this approach. Through the examination of structural properties of the different residues, it was explored for the first time that lysine is not the only responsible of the denaturation mechanism. Based on the simulation results, the denaturation process occurs by escaping of many surface charged and polar residues from the solvent and forming non-native H bonds with the other residues of the enzyme in a cooperative process. This changes the interaction pattern of the enzyme residues in supercritical CO2. In fact, the denaturation process is mainly due to the many new non-native H bonds of surface charged and polar residues and is not a result of single residue instability. The new interactions lead to the destruction of many essential native H bonds of the enzyme. We have evaluated this mechanism by simulations of lipase and peroxidase as two enzymes with the different activity and stability in supercritical CO2. Structural analysis, solvent accessible surface area, and H bond numbers confirm that both enzymes show similar behavior to lysozyme in supercritical CO2, although with the different extents. Lipase showed lower change of solvent accessible surface area and fewer new H bonds in comparison to peroxidase, which are in line with the experimental observations of more stability and activity of this enzyme in comparison to peroxidase in supercritical CO2. Moreover, both enzymes show normal behavior with minimal changes and new interactions in the aqueous conditions. We think that this new mechanism can explain the reasons of enzyme denaturation and justify the different behaviors of the enzymes in supercritical CO2 solvent. It was also revealed that creating more interactions among the polar and charged residues at the enzyme surface and increasing the surface hydrophobicity could be effective ways in stabilizing the enzymes in supercritical CO2.

Molecular Modeling Investigation of the Interaction between Humicola insolens Cutinase and SDS Surfactant Suggests a Mechanism for Enzyme Inactivation.

One of the largest commercial applications of enzymes and surfactants is as main components in modern detergents. The high concentration of surfactant compounds usually present in detergents can, however, negatively affect the enzymatic activity. To remedy this drawback, it is of great importance to characterize the interaction between the enzyme and the surfactant molecules at an atomistic resolution. The protein enzyme cutinase from the thermophilic and saprophytic fungus called Humicola insolens (HiC) is a promising candidate for use in detergents thanks to its hydrolase activity targeting mostly biopolyesters (e.g., cutin). HiC is, however, inhibited by low concentrations of sodium dodecyl sulfate (SDS), an ubiquitous surfactant. In this work, we investigate the interaction between HiC and SDS using molecular dynamics simulations. Simulations of HiC dissolved in different aqueous concentrations of SDS show the interaction between HiC and SDS monomers, as well as the formation and dynamics of SDS micelles on the surface of the enzyme. These results suggest a mechanism of cutinase inhibition by SDS, which involves the nucleation of aggregates of SDS molecules on hydrophobic patches on the cutinase surface. Notably, a primary binding site for monomeric SDS is identified near the active site of HiC constituting a possible nucleation point for micelles and leading to the blockage of the entrance to the enzymatic site. Detailed analysis of the simulations allow us to suggest a set of residues from the SDS binding site on HiC to probe as engineered mutations aimed at reducing SDS binding to HiC, thereby decreasing SDS inhibition of HiC.

Molecular mechanism of enzyme tolerance against organic solvents: Insights from molecular dynamics simulation

Biocatalysis in presence of organic solvents has numerous industrially attractive advantages in comparison to traditional aqueous solvents. In some cases, the presence of organic molecules such as methanol in the processes such as enzymatic production of biodiesel is inevitable. However, enzyme inactivation and/or instability in organic solvents limits such biotechnological processes. Although it was found that some enzymes are more and others are less tolerant against organic solvents, the structural basis of such differences is relatively unknown. In this work, using molecular dynamics simulations, we have investigated the structural behavior of enzymes with completely different structural architecture including lipase, laccase and lysozyme in the presence of methanol as polar and hexane as non-polar organic solvents. In agreement with the previous experimental observations, simulations showed that lipase is more tolerant against both polar and non-polar organic solvents. It is found that lipase has high stability in pure hexane even higher than that obtained in the aqueous solvent. In contrast, laccase shows better stability in the aqueous conditions. To obtain general mechanism of enzyme inactivation in the presence of methanol and hexane, we have treated lysozyme as model enzyme in the different percentages of these solvents in long MD simulations. It is found that lysozyme is completely denatured at high concentration- of methanol, but it remains native at low concentration of this solvent. Interestingly, the concentration-dependence structural behavior of enzyme was completely different in the presence of hexane. It was obtained that low concentrations of hexane may impose more instability on the enzyme conformation than higher percentages. Results also showed that presence of water is determining factor in the enzyme stability at high concentrations of hexane. Pure hexane may also lead to the surface denaturation of the enzymes. Both methanol and hexane denaturation mechanisms were initiated by diffusion of organic solvent in hydrophobic core. However, enzyme denaturation in hexane was continued by a collapse of hydrophobic core and entering hexane molecules to the core, but in methanol it was completed by decomposition of the secondary structures. In both cases it was found that beta structures are more prone to destabilize than helix structures. This may be a reason for obtained results about lower stability of laccase with β-barrel architecture than lipase with multiple helixes at it surface. In total, by our extensive structural data, it was found that the forces which stabilize tertiary structure have pivotal role in enzyme tolerance against both polar and non-polar organic solvents.

Insight into the mode of action and selectivity of PBRM, a covalent steroidal inhibitor of 17β-hydroxysteroid dehydrogenase type 1

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is involved in the biosynthesis of estradiol, the major bioactive endogenous estrogen in mammals, and constitutes an interesting therapeutic target for estrogen-dependent diseases. A steroidal derivative, 3-{[(16β,17β)-3-(2-bromoethyl)-17-hydroxyestra-1,3,5(10)-trien-16-yl]methyl} benzamide (PBRM), has recently been described as a non-estrogenic, irreversible inhibitor of 17β-HSD1. However, the mode of action of this inhibitor and its selectivity profile have not yet been elucidated. We assessed PBRM potency via in vitro kinetic measurements. The mechanism of enzyme inactivation was also investigated using interspecies (human, mouse, pig and monkey) comparisons via both in vitro assays and in silico analysis. Mouse and human plasma protein binding of PBRM was determined, whereas its selectivity of action was studied using a wide range of potential off-targets (e.g. GPCR, hERG, CYPs, etc.). The affinity constant (Ki=368nM) and the enzyme inactivation rate (kinact=0.087min−1) values for PBRM were determined with purified 17β-HSD1. PBRM was found to be covalently linked to the enzyme. A long delay period (i.e. 3–5days) is required to recover 17β-HSD1 activity following a pretreatment of breast and placenta cell lines with PBRM. Mechanistic analyses showed important interspecies differences of 17β-HSD1 inhibition which support the importance of inactivation for PBRM effect. Evidences of the potency and selectivity of action presented herein for this first non-estrogenic and steroidal covalent irreversible inhibitor of 17β-HSD1 warrant its further development as a potential drug candidate for estrogen-dependent disorders.

Thermal inactivation kinetics of β-galactosidase during bread baking.

In this study, β-galactosidase was utilized as a model enzyme to investigate the mechanism of enzyme inactivation during bread baking. Thermal inactivation of β-galactosidase was investigated in a wheat flour/water system at varying temperature-moisture content combinations, and in bread during baking at 175 or 205°C. In the wheat flour/water system, the thermostability of β-galactosidase increased with decreased moisture content, and a kinetic model was accurately fitted to the corresponding inactivation data (R2=0.99). Interestingly, the residual enzyme activity in the bread crust (about 30%) was hundredfold higher than that in the crumb (about 0.3%) after baking, despite the higher temperature in the crust throughout baking. This result suggested that the reduced moisture content in the crust increased the thermostability of the enzyme. Subsequently, the kinetic model reasonably predicted the enzyme inactivation in the crumb using the same parameters derived from the wheat flour/water system. However, the model predicted a lower residual enzyme activity in the crust compared with the experimental result, which indicated that the structure of the crust may influence the enzyme inactivation mechanism during baking. The results reported can provide a quantitative understanding of the thermal inactivation kinetics of enzyme during baking, which is essential to better retain enzymatic activity in bakery products supplemented with heat-sensitive enzymes.

Investigation of structural stability and enzymatic activity of glucose oxidase and its subunits

Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) catalyzes the oxidation of β-d glucose utilizing molecular oxygen as an electron acceptor to produce d-glucono-1,5-lactoneand hydrogen peroxide, which has applications in food, biotechnology and medical industries. It was known that dimer form was considered to be active and monomer form has inactive conformation. However, there are no evidences at the molecular levels for Glucose oxidase (GOx) inactivation through dissociation. Here, using molecular dynamic simulation, it has been investigated for the first time that why dimer form of the enzyme is active. We have performed a series of molecular dynamics simulations at different forms of GOx (monomer and dimer with and without FAD cofactor). The analysis of tertiary structure showed that monomer is more unstable and has more deviation from the crystal structure. In contrast, dimer has a stable conformation during simulation. These results are in good agreement with experimental data about enzyme inactivation by dissociation. It was also found that when FAD is removed from monomer, it became more unstable in comparison with monomer containing cofactor. This shows essential role of FAD in both activity and stability of the enzyme. According to the MD simulation, enzyme inactivation is associated with changing in secondary structure at the interface. Interestingly, it was found that some secondary structures are destructed while some structures are formed in monomer upon dissociation. The analysis of active site structure during simulation revealed that both dissociation and release of the FAD influence on inactivation of GOx. This study provided novel insight to understand the mechanism of enzyme inactivation upon dissociation, which would be useful for rational enzyme design.

Chemical modification of biocatalyst for function in supercritical CO2: In silico redesign of stable lipase

In spite of excellent properties of supercritical CO2 in enzyme catalyzed reactions, destabilizing effects of CO2 molecules on the enzyme structure limits the industrial applications of this green solvent in the field of biocatalysis. Here, based on the substantial role of charged surface residues such as lysines in enzyme inactivation, we introduced for the first time, Post Translational Modifications (PTMs), a famous concept in molecular biology, as a protein stabilization strategy in supercritical condition. Lysine groups were modified using PTM templates to find out more about the exact mechanism of enzyme inactivation in supercritical CO2 and to explore a new way for protein stabilization in this solvent. We used MD simulation as common tool for in situ examining enzyme structure in supercritical fluids, for the investigation of structural consequence of modifications. Different modifications including acetylation, methylation, phosphorylation, and carboxylation have been applied on the model enzyme. For comparison to CALB structure in supercritical CO2, the acetylated enzyme was also simulated in aqueous solvent at 300 and 353K. Interestingly, acetylation of lysine residues prevents enzyme from denaturation at high temperatures in water, which is in agreement with experimental observation in aqueous solution. However, acetylation is not so useful for stabilization of enzyme in supercritical CO2. In contrast, methylation and carboxylation efficiently stabilize enzyme in supercritical CO2. Phosphate groups in phosphorylated lysines destabilize enzyme by formation of excess hydrogen bonds by inappropriate groups and perturb enzyme conformation. Moreover, it was found that modification of surface arginine residues was not so satisfactory in stabilization of the enzyme. This finding supports the mechanism of lipase inactivation through direct interaction of CO2 molecules on lysine residues and formation of carbamates. We think this new exploration can open new window for developing enzyme catalyzed mechanism of enzyme inactivation in scCO2.

Thermal versus Microwave Inactivation Kinetics of Lipase and Lipoxygenase from Wheat Germ

AbstractThe inactivation kinetics of enzymes is an important way to reveal the mechanism of enzyme inactivation by microwaves. In this paper, lipase (LA) and lipoxygenase (LOX) were treated by both microwave irradiation and conventional heating. A temperature‐controlled water bath was used for conventional heating, and a microwave monomode reactor with a simultaneous cooling and precise temperature control system was used for microwave irradiation. The results showed that the effects of both microwave irradiation and conventional heating on enzyme inactivation were enhanced as system temperature increased; LOX was more sensitive to heat than LA. Therefore, compared with conventional heating, microwave irradiation shows no significant differences in inactivating LA or LOX in aqueous solution, and the thermal effect of microwave irradiation is the main cause of enzyme inactivation.Practical ApplicationsWheat germ has a short shelf life because of the high activity of endogenous enzymes. Two relevant endogenous enzymes are involved in the degradation of nutrients, lipase (LA) and lipoxygenase (LOX); therefore, the inactivation of LA and LOX is an essential prerequisite for long‐term storage of wheat germ. Microwave irradiation treatment (MIT) has long been applied to the inactivation of enzymes and has several advantages such as having a minimal effect on food product quality and providing rapid and uniform heating. However, the mechanism of enzyme inactivation by microwaves is still controversial. In this study, wheat germ endogenous enzymes were treated by MIT and conventional heat treatment. Based on residual enzyme activities and first‐order rate of inactivation kinetics, the mechanism of microwave inactivation was demonstrated, offering a theoretical basis for the future application of this technique in grain stabilization.

High-Pressure Inactivation of Enzymes: A Review on Its Recent Applications on Fruit Purees and Juices.

In the last 2 decades high-pressure processing (HPP) has established itself as one of the most suitable nonthermal technologies applied to fruit products for the extension of shelf-life. Several oxidative and pectic enzymes are responsible for deterioration in color, flavor, and texture in fruit purees and juices (FP&J). The effect of HPP on the activities of polyphenoloxidase, peroxidase, β-glucosidase, pectinmethylesterase, polygalacturonase, lipoxygenase, amylase, and hydroperoxide lyase specific to FP&J have been studied by several researchers. In most of the cases, partial inactivation of the target enzymes was possible under the experimental domain, although their pressure sensitivity largely depended on the origin and their microenvironmental condition. The variable sensitivity of different enzymes also reflects on their kinetics. Several empirical models have been established to describe the kinetics of an enzyme specific to a FP&J. The scientific literature in the last decade illustrating the effects of HPP on enzymes in FP&J, enzymatic action on those products, mechanism of enzyme inactivation during high pressure, their inactivation kinetics, and several intrinsic and extrinsic factors influencing the efficacy of HPP is critically reviewed in this article. In addition, process optimization of HPP targeting specific enzymes is of great interest from an industrial approach. This review will give a fair idea about the target enzymes specific to FP&J and the optimum conditions needed to achieve sufficient inactivation during HPP treatment.

The improvement of stability, activity, and substrate promiscuity of glycerol dehydrogenase substituted by divalent metal ions

The substitution of the catalytic zinc ion of glycerol dehydrogenase (GDH) from Klebsiella pneumonia sp. by divalent metal ions, Mn2+ and Mg2+, enabled improvements of activity, substrate promiscuity and stability. The activity of Mn-GDH and Mg-GDH improved several folds in comparison to the native GDH. The activity of substituted GDH towards non-natural substrates, 4-chloroacetoacetate, 3-chloroacetylpyridine, p-chloroacetophenone, and acetophenone was 30 folds higher than native GDH. Manganese substitution increased the half-life of GDH by 6 folds at 60 and 70°C. The two-fraction first order inactivation models fitted the nonlinear thermal inactivation curves well. Combined with the kinetic and thermodynamic analysis, further mechanistic insights to the metal ion roles in thermostability enhancements were studied. The thermodynamic parameters of inactivation, enthalpy, entropy and the Gibbs free energy indicated that Mn-GDH was stabilized entropically and elucidated the mechanisms of enzyme inactivation.

Inactivation of Glucosamine‐6‐Phosphate Synthase by N3‐Oxoacyl Derivatives of L‐2,3‐Diaminopropanoic Acid

N(3)-Oxoacyl derivatives of L-2,3-diaminopropanoic acid 1-4, containing either an epoxide group or a conjugated double bond system, inactivate Saccharomyces cerevisiae glucosamine-6-phosphate (GlcN-6-P) synthase in a time- and concentration dependent manner. The results of kinetics studies on inactivation suggested a biphasic course, with formation of the enzyme-ligand complex preceding irreversible modification of the enzyme. The examined compounds differed markedly in their affinity to the enzyme active site. Inhibitors containing a phenyl ketone moiety bound much more strongly than their methyl ketone counterparts. The molecular mechanism of enzyme inactivation by phenyl ketone compounds 1 and 3 was elucidated by using a stepwise approach with 2D NMR, MS and UV-visible spectroscopy. A substituted thiazine derivative was identified as the final product of a model reaction between an epoxide compound, 1, and L-cysteine ethyl ester (CEE); and the respective cyclic product, found as a result of reaction between 1 and CGIF tetrapeptide, was identical to the N-terminal fragment of GlcN-6-P synthase. On the other hand, the reaction of a double-bond-containing compound, 3, with CEE, CGIF and GlcN-6-P synthase led to the formation of a C-S bond, without any further conversion or rearrangement. Molecular mechanisms of the reactions studied are proposed.

Fatty Acid Amide Hydrolase Inhibitors – Progress and Potential

Fatty acid amide hydrolase (FAAH) is responsible for hydrolysis of endocannabinoid, anandamide (AEA), and N-acyl ethanolamines such as palmitoylethanolamine (PEA) and N-oleoylethanolamide (OEA). Genetic deletion or pharmacological inactivation of FAAH shows site-specific elevation of AEA that plays a role in the modulation of pain and other neurodegenerative disorders. The review elaborates recent progress and current status of diverse structural classes of reversible and irreversible FAAH inhibitors. The discussion also addresses ligand-enzyme active site interactions and mechanism of enzyme inactivation, emerging approaches to novel FAAH inhibitors, and ongoing efforts to address gaps in therapeutic utility of FAAH inhibitors.

Pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde–alcohol dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba histolytica

The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O(2) exposure (IC(50) 0.034 mm), whereas short-term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe(2+). CoA and acetyl-CoA prevented, whereas pyruvate exacerbated, inactivation induced by short-term saturating O(2) exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe(2+)-reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe-S clusters. After O(2) exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde-alcohol dehydrogenase (EhADH2), whereas acetyl-CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of EhADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress.

Characterization of site-specific mutants of alkylhydroperoxide reductase with dual functionality from Helicobacter pylori

Alkylhydroperoxide reductase (AhpC) is an abundant and important antioxidant protein present in Helicobacter pylori (HP), a spiral Gram-negative microaerophilic bacterium. By sequence alignment and structure comparison, HP-AhpC was found to be more homologous to human peroxiredoxins (hPrx) than to other eubacterial AhpC proteins. Similar to hPrxI, native HP-AhpC existed as a dimer of single subunit, comprising alpha-helix and beta-sheet domains with low surface hydrophobicity. AhpC can form high-molecular-weight (HMW) aggregates ranging from 700 to higher than 2,000 kDa under oxidative stress, possessing chaperone activity in the presence of thioredoxin (Trx). Further analysis of peroxide-reductase activities showed that HP-AhpC was more resistant to H(2)O(2) than hPrxI. However, the mechanism of enzyme inactivation to H(2)O(2) appeared to be similar for both HP-AhpC and hPrxI as revealed by native gel electrophoresis followed by proteomic identification using two-dimensional gel electrophoresis (2-DE) and LC-MS/MS. In contrast to T90D-hPrxI mutant with chaperone activity, site-specific mutant T87D-HP-AhpC did not form HMW chaperone complexes. The comparison of these two evolutionarily distant and yet functionally related enzymes may shed some light on the mechanism(s) underlying the evolution and development of the dual functionality in HP-AhpC and hPrxI with similar protein structure.

How algae produce hydrogen—news from the photosynthetic hydrogenase

Green algae are the only known eukaryotes capable of oxygenic photosynthesis which are equipped with a hydrogen metabolism. Hydrogen production is light-dependent, since the [FeFe] hydrogenases are coupled to the photosynthetic electron transport chain via ferredoxin. Algal [FeFe] hydrogenases are one of the most active biocatalysts for the evolution of hydrogen. Therefore, special interest exists in the biophysical characterization and biotechnological usage of these [Fe-S] enzymes. This review traces the discovery of this interesting class of proteins. Recent findings allow insight into the electronic structure and configuration of the [FeFe] hydrogenase active site (H-cluster). Emphasis is placed on novel discoveries of the hydrogenase interaction with its natural electron donor ferredoxin and the mechanism of enzyme inactivation through oxygen.

Radiofrequency inactivation of oxidative food enzymes in model systems and apple derivatives

Abstract The effect of increasing radiofrequency (RF) electric fields on the activity of oxidative enzymes was studied in model systems and food. In particular, the mechanisms of enzyme inactivation by RF treatments were studied on polyphenoloxidase (PPO) and lipoxygenase (LOX) model systems. RF treatments efficiently inactivated both enzymes, although PPO was more sensitive to an increase in the RF electric field. RF efficacy was attributed to the generated thermal effect, while the contribution of the electromagnetic field resulted negligible. Following, the efficacy of RF in blanching vegetables to obtain color stable derivatives was evaluated. To this aim, RF and conventionally water blanched apples were processed to get purees which were analyzed for color, sensory attributes and consumer preference. RF allowed apples to be adequately blanched. In particular, the puree obtained from the RF blanched apples was judged comparable to the conventionally water blanched one for color and sensory attributes. However, a slightly higher perceivable sweetness of the RF blanched sample was found, probably due to the fact that the RF treatment allowed any contact with water to be avoided.

Surface characterization and direct electrochemistry of redox copper centers of bilirubin oxidase from fungi Myrothecium verrucaria

The key characteristics of multicopper oxidases are redox potentials of Type 1, Type 2 and Type 3 copper centers of enzymes. However, there is still a challenge to obtain a value of the redox “signature” of the enzymes. In this study, the electrochemical behavior of T1 and T2/T3 redox copper centers of bilirubin oxidase (BOD) from the fungi Myrothecium verrucaria was studied based on direct bioelectrocatalysis. Two distinct redox peaks corresponding to reduction and oxidation of T1 and T2/T3 redox centers of enzymes have been clearly detected in anaerobic conditions. The bioelectrocatalytic activity of the enzyme was studied in the presence of oxygen and redox mediators. The electron-transfer rate constant for BOD immobilized on carbon electrode (CE) is 1.5 s−1. The mechanism of enzyme inactivation by ABTS has been proposed. The physical architecture of BOD layers immobilized on the electrode surface, including elemental and chemical composition, relative thickness and assembly of layers was investigated by Angle Resolved X-ray photoelectron spectroscopy. Unique peaks of BOD at 288.5 eV and of CE at 284.6 eV were used in a substrate over layer model for estimation of the thickness of the of BOD film on the carbon electrode surface.

Pyridoxal 5-Phosphate Enzymes as Targets for Therapeutic Agents

The vitamin B(6)-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in amino acid metabolism. These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and protein structural organization. Almost 1.5% of all genes in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of enzyme-catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), serine hydroxymethyltransferase (tumors and malaria), ornithine decarboxylase (African sleeping sickness and, potentially, tumors), alanine racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or metabolic pathway, the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and on-going research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.