Considerations about the Continuous Assay Methods, Spectrophotometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase

With the purpose to obtain the more useful tyrosinase assay for the monophenolase activity of tyrosinase between the spectrofluorometric and spectrophotometric continuous assays, simulated assays were made by means of numerical integration of the equations that characterize the mechanism of monophenolase activity. These assays showed that the rate of disappearance of monophenol (VssM,M) is equal to the rate of accumulation of dopachrome (VssM,DC) or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on o-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (VssM, A−ox), despite the existence of coupled reactions. It is shown that the spectrophotometric methods that use MBTH are more useful, as they do not have the restrictions of the L-tyrosine disappearance measurement method, of working at pH = 8 and not having a linear response from 100 μM of L-tyrosine. It is possible to obtain low LODM (limit of detection of the monophenolase activity) values with spectrophotometric methods. The spectrofluorimetric methods had a lower LODM than spectrophotometric methods. In the case of 4-hydroxyphenil-propionic acid, the LODM obtained by us was 0.25 U/mL. Considering the relative sensitivities of 4-hydroxyanisole, compared with 4-hydroxyphenil-propionic acid, LODM values like those obtained by fluorescent methods would be expected.

N6-methyladenosine binding induces a metal-centered rearrangement that activates the human RNA demethylase Alkbh5

Alkbh5 catalyzes demethylation of the N6-methyladenosine (m6A), an epigenetic mark that controls several physiological processes including carcinogenesis and stem cell differentiation. The activity of Alkbh5 comprises two coupled reactions. The first reaction involves decarboxylation of α-ketoglutarate (αKG) and formation of a Fe4+═O species. This oxyferryl intermediate oxidizes the m6A to reestablish the canonical base. Despite coupling between the two reactions being required for the correct Alkbh5 functioning, the mechanisms linking dioxygen activation to m6A binding are not fully understood. Here, we use solution NMR to investigate the structure and dynamics of apo and holo Alkbh5. We show that binding of m6A to Alkbh5 induces a metal-centered rearrangement of αKG that increases the exposed area of the metal, making it available for binding O2. Our study reveals the molecular mechanisms underlying activation of Alkbh5, therefore opening new perspectives for the design of novel strategies to control gene expression and cancer progression.

Thermodynamics drives coenzyme redundancy in metabolism

Coenzymes redistribute a variety of resources (e.g., electrons, phosphate groups, methyl groups) throughout cellular metabolism. For a variety of reactions requiring acceptors or donors of specific resources, there often exist degenerate sets of molecules (e.g., NAD(H) and NADP(H)) that carry out similar functions. Several hypotheses can explain the persistence of coenzyme degeneracy, but none have been tested quantitatively. Here, we use genome-wide metabolic modeling approaches to decompose the selective pressures driving enzymatic specificity for coenzymes in the metabolic network of Escherichia coli. Flux balance modeling predicts that only two enzymes (encoded by leuB and pdxB) are thermodynamically constrained to use NAD(H) over NADP(H). In contrast, structural and sequence analyses reveal widespread conservation of residues that retain selectivity for NAD(H), suggesting that additional forces drive enzyme specificity. Using a model accounting for the cost of oxidoreductase enzyme expression, we find that coenzyme redundancy universally reduces the minimal amount of protein required to catalyze coenzyme-coupled reactions, inducing individual reactions to strongly prefer one coenzyme over another when reactions are near thermodynamic equilibrium. We propose that partitioning of flux across multiple coenzyme pools could be a generic phenomenon of cellular metabolism, and hypothesize that coenzymes typically thought to exist in a single pool (e.g., CoA) may exist in more than one form (e.g., dephospho-CoA).

Influence of Slag Composition on the Distribution Behavior of Cu between Liquid Sulfide and Cu-Containing Multicomponent Slag via Thermodynamic and Kinetic Assessment

At present, copper smelting slag is not effectively recycled and is wasted. Copper smelting slag contains FexO at more than 40 mass%. For the utilization of copper slag as a Fe resource, it is necessary to separate the Cu in the slag. For copper recycling from slag, FeS-based matte can be introduced to use sulfurization to concentrate Cu from the slag into the sulfide and finally recover the copper. In a previous paper, a kinetic model was developed to simulate the coupled reactions between the multicomponent slag and FeS-based matte by using previously reported thermodynamic data. Building on this work, we carried out equilibrium experiments to supplement the thermodynamic data used in the previously developed model. An empirical formula for the Cu2O activity coefficient of Cu2O-FeOX-CaO-MgO-SiO2-Al2O3 system slag was obtained. In addition, the effect of alumina content in the slag on the Cu2O activity coefficient in the slag was investigated. The model was also supplemented to account for MgO solubility. By the developed model and the industrial conditions, we investigated the effect of slag composition on the behavior of Cu between matte and Cu2O-FeOX-CaO-MgO-SiO2-Al2O3 system slag for the copper loss.

Toughening Hydrogels Through Force-triggered Chemical Reactions that Lengthen Polymer Strands

<p>The utility and lifetime of materials made from polymer networks, including hydrogels, depend on their capacity to stretch and resist tearing. In gels and elastomers, those mechanical properties are often limited by the covalent chemical structure of the polymer strands between cross-links, which is typically fixed during the material synthesis. Here, we report polymer networks in which the constituent strands lengthen through force-coupled reactions that are triggered as the strands reach their nominal breaking point. Reactive strand extensions of up to 40% lead to hydrogels that stretch 40-50% further than, and exhibit tear energies twice that of, networks made from analogous control strands. The enhancements are synergistic with those provided by double network architectures, and complement other existing toughening strategies. </p>

Toughening Hydrogels Through Force-triggered Chemical Reactions that Lengthen Polymer Strands

<p>The utility and lifetime of materials made from polymer networks, including hydrogels, depend on their capacity to stretch and resist tearing. In gels and elastomers, those mechanical properties are often limited by the covalent chemical structure of the polymer strands between cross-links, which is typically fixed during the material synthesis. Here, we report polymer networks in which the constituent strands lengthen through force-coupled reactions that are triggered as the strands reach their nominal breaking point. Reactive strand extensions of up to 40% lead to hydrogels that stretch 40-50% further than, and exhibit tear energies twice that of, networks made from analogous control strands. The enhancements are synergistic with those provided by double network architectures, and complement other existing toughening strategies. </p>