Inactivation of Choline Oxidase by Irreversible Inhibitors or Storage Conditions

Choline oxidase from Arthrobacter globiformis is a flavin-dependent enzyme that catalyzes the oxidation of choline to betaine aldehyde through two sequential hydride-transfer steps. The study of this enzyme is of importance to the understanding of glycine betaine biosynthesis found in pathogenic bacterial or economic relevant crop plants as a response to temperature and salt stress in adverse environment. In this study, chemical modification of choline oxidase using two irreversible inhibitors, tetranitromethane and phenylhydrazine, was performed in order to gain insights into the active site structure of the enzyme. Choline oxidase can also be inactivated irreversibly by freezing in 20 mM sodium phosphate and 20 mM sodium pyrophosphate at pH 6 and -20 oC. The results showed that enzyme inactivation was due to a localized conformational change associated with the ionization of a group in close proximity to the flavin cofactor and led to a complete lost of catalytic activity

The impact of adsorbed cellulase inactivation on enzymatic hydrolysis kinetics.

Several technical and economic obstacles currently hamper the industrial development of ethanol from biomass. One of the key bottlenecks is the slow kinetics of the enzymatic hydrolysis of cellulose, and the subsequent rate reduction as the reaction proceeds. As a result, this research focused on understanding underlying causes for the slow kinetics, rate reduction, and low yield during cellulose hydrolysis. Mechanisms traditionally thought to cause these results were investigated, such as change of substrate properties and deactivation of enzyme due to environmental mechanisms, but neither was found to contribute significantly to the slow kinetics and low yield. Inactivation due to enzyme-substrate interactions was then proposed as a key factor. Results here show that inactivation of adsorbed enzyme played the most significant role for the hydrolysis rate reduction and low yield based on the following findings: (1) a kinetic model featuring inactivation of adsorbed enzyme accurately accounted for experimental cellulose hydrolysis data for two different types of substrates; the enzyme\u27s apparent maximum reaction rate was found to decrease with a first order exponential decay function of time due to inactivation of the adsorbed enzyme, which has historically always been considered to remain constant. (2) comparison of relative extents of enzyme activity loss due to environmental mechanisms (such as thermal and/or mechanical factors) with inactivation due to enzyme-substrate interactions revealed that enzyme- substrate interactions contributed more towards the overall activity loss than did environmental mechanisms; (3) AFM imaging visualized crowding of Cellobiohydrolase 1 (CBHl) on cellulose substrate surface and thereafter became inactivated; (4) desorption of inactive CBHl was slower compared to desorption of active CBHl, implying that once inactivated, CBH 1 cannot dissociate immediately to find another site on a substrate surface to start another digestive cycle. The overall conclusion is that inactivation of adsorbed enzyme is a primary contributor to the hydrolysis rate reduction. Near complete conversion (99%) of cellulose was predicted by the model to occur within 10-20 hours if inactivation of adsorbed cellulase can be prevented, compared to 7-10 days or more to achieve a lower yield when inactivation occurs. Finally, factors to consider when developing a cellulose hydrolysis process were proposed based on the inactivation mechanism. One important strategy proposed is to desorb inactive cellulases from the substrate, such as with the addition of GdnHCl. Additionally, a technique for scaling-up separation of CBHl was developed. The technique allows for efficient purification of active CBHl from commercial cellulose cocktails at a cost of less than 10% compared to the conventional small-scale FPLC method

Inhibitors of Pyruvate Carboxylase

This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate

Soybean peroxidase catalyzed polymerization and removal of 2,4-dimethylphenol from synthetic wastewater

Enzymatic treatment of synthetic wastewater containing 2,4-dimethylphenol (2,4-DMP) was investigated in the presence and absence of polyethylene glycol (PEG) by the enzyme soybean peroxidase. The optimum pH both in the absence and in presence of PEG was 8.0. The optimum [hydrogen peroxide]/[2,4-DMP] was between 0.9-1.2. A linear relationship existed in presence of PEG between the minimum SBP concentration and initial 2,4-DMP concentrations. In the absence of PEG, a linear relationship did exist at lower substrate concentrations up to 2.0 mM, beyond which the minimum enzyme concentration remained constant and independent of the initial substrate concentration. At lower 2,4-DMP concentrations, there was PEG effect which decreased to almost nil with increase in substrate concentrations. Minimum PEG concentration for 1 mM of 2,4-DMP was found to be 45-50 mg/L. Preliminary kinetic study of the enzyme-catalyzed reaction yielded the values of Michaelis-Menten constants for 2,4-dimethylphenol, in the presence and absence of PEG

In Silico screening of nonsteroidal anti-inflammatory drugs and their combined action on Prostaglandin H Synthase-1

The detailed kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was applied to in silico screening of dose-dependencies for the different types of nonsteroidal anti-inflammatory drugs (NSAIDs), such as: reversible/irreversible, nonselective/selective to PGHS-1/PGHS-2 and time dependent/independent inhibitors (aspirin, ibuprofen, celecoxib, etc.) The computational screening has shown a significant variability in the IC50s of the same drug, depending on different in vitro and in vivo experimental conditions. To study this high heterogeneity in the inhibitory effects of NSAIDs, we have developed an in silico approach to evaluate NSAID action on targets under different PGHS-1 microenvironmental conditions, such as arachidonic acid, reducing cofactor, and peroxide concentrations. The designed technique permits translating the drug IC50, obtained in one experimental setting to another, and predicts in vivo inhibitory effects based on the relevant in vitro data. For the aspirin case, we elucidated the mechanism underlying the enhancement and reduction (aspirin resistance) of its efficacy, depending on PGHS-1 microenvironment in in vitro/in vivo experimental settings. We also present the results of the in silico screening of the combined action of sets of two NSAIDs (aspirin with ibuprofen, aspirin with celecoxib), and study the mechanism of the experimentally observed effect of the suppression of aspirin-mediated PGHS-1 inhibition by selective and nonselective NSAIDs. Furthermore, we discuss the applications of the obtained results to the problems of standardization of NSAID test assay, dependence of the NSAID efficacy on cellular environment of PGHS-1, drug resistance, and NSAID combination therapy

On the Biochemistry, Mechanism and Physiological Role of Fungal Nitronate Monooxygenase

Nitronate monooxygenase (E.C. 1.13.11.16), formerly known as 2-nitropropane dioxygenase (EC 1.13.11.32), is a flavin dependent enzyme that catalyzes the oxidation of nitronates to their corresponding carbonyl compounds and nitrite. Despite the fact that the enzyme was first isolated from Neurospora crassa 60 years ago, the biochemical and physiological properties of nitronate monooxygenase have remained largely elusive. This dissertation will present the work that established both the catalytic mechanism and physiological role of the fungal enzyme. The biological and biochemical properties of propionate-3-nitronate, the recently discovered physiological substrate for nitronate monooxygenase, will be extensively reviewed. The nitronate is produced by a variety of variety leguminous plants and fungi and is a potent and irreversible inhibitor of succinate dehydrogenase. Nitronate monooxygenase allows N. crassa to overcome the toxicity of propionate-3-nitronate as demonstrated by in vivo studies of the yeast, which showed that the wild-type can grow in the presence of the toxin whereas a knock out mutant that lacks the gene encoding for the enzyme could not. In addition to establishing the physiological role of nitronate monooxygenase, the work presented here demonstrates that the catalytic mechanism of the enzyme involves the formation of an anionic flavosemiquinone intermediate. This intermediate is stabilized by the protonated form of an active site histidine residue (His-196) that acts as an electrostatic catalyst for the reaction as demonstrated by pH studies of the reductive half reaction of the enzyme. Histidine 196 also serves as the catalytic base for the reaction of the enzyme with nitroethane as substrate as revealed through mutagenesis studies in which the residue was replaced with an asparagine. The kinetic implications of branching of reaction intermediates in enzymatic catalysis are also demonstrated through studies of the kinetic isotope effects of nitronate monooxygenase with 1,1-[2H2]-nitroethane as substrate. Finally the use of competitive inhibitors as a probe of enzyme structure will be presented through a study of the inhibition of nitronate monooxygenase with mono-valent inorganic ions. The dissertation will close with unpublished work on the enzyme and concluding remarks concerning the biochemistry and physiology of nitronate monooxygenase

Functional characterization and structure-guided mutational analysis of the transsulfuration enzyme cystathionine γ-lyase from toxoplasma gondii

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine β-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of T. gondii. However, their functionality has not been investigated. Herein, we expressed and characterized the putative CGL from T. gondii. Recombinant TgCGL almost exclusively catalyses the α,γ-hydrolysis of L-cystathionine to form L-cysteine and displays marginal reactivity toward L-cysteine. Structure-guided homology modelling revealed two striking amino acid differences between the human and parasite CGL active-sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of Asn360 to Ser demonstrated the importance of this residue in modulating the specificity for the catalysis of α,β-versus α,γ-elimination of L-cystathionine. Replacement of Ser77 by Glu completely abolished activity towards L-cystathionine. Our results suggest that CGL is an important functional enzyme in T. gondii, likely implying that the reverse transsulfuration pathway is operative in the parasite; we also probed the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsulfuration enzymes