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The flavoenzyme glycolate oxidase oxidizes glycolic acid to glyoxylate and the latter, more slowly, to oxalate. It is a member of an FMN‐dependent enzyme family that oxidizes l‐2‐hydroxy acids to keto acids. There has been a controversy concerning the chemical mechanism of substrate oxidation by these enzymes. Do they proceed by hydride transfer, as observed for NAD‐dependent enzymes, or by initial formation of a carbanion that transfers the electrons to the flavin? The present work describes a comparison of the reactivity of glycolate, lactate and trifluorolactate with recombinant human glycolate oxidase, by means of rapid‐kinetics experiments in anaerobiosis. We show that trifluorolactate is a substrate for glycolate oxidase, whereas it is known as an inhibitor for NAD‐dependent enzymes, as is trifluoroethanol for NAD‐dependent alcohol dehydrogenases. Unexpectedly, it was observed that, once reduced, a flavin transfers an electron to an oxidized flavin, so that the end species is a flavin semiquinone, whatever the substrate. This phenomenon has not previously been described for a glycolate oxidase. Altogether, considering that another member of this flavoenzyme family (flavocytochrome b2, a lactate dehydrogenase) has also been shown to oxidize trifluorolactate (Lederer F et al. (2016) Biochim Biophys Acta 1864, 1215–21), this work provides another important piece of evidence which is hardly compatible with a hydride transfer mechanism for this flavoenzyme family. [ABSTRACT FROM AUTHOR]

The mechanism of Saccharomyces cerevisiae flavodehydrogenase domain is reexamined to study its properties. Results indicate that flavin-reduction is the limiting step in lactate oxidation.

Research has been conducted on the Saccharomyces cerevisiae flavocytochrome b (sub)2. The investigation of the crystal structure of the yeast enzyme has been carried out.

A study was conducted to analyze and characterize the production of monoclonal antibodies against flavocytochrome b2. The flavocytochromes b2 were purified and the kinetic measurements of the enzymic oxidation were performed. Results revealed that monoclonal antibodies recognized the heme-binding domain and the mobility of the cytochrome domain of the flavocytochrome b2 was essential to catalytic cycle.

Stopped-flow and steady-state studies provided the detailed investigation for the inhibition of both cytochrome c and ferricyanide reduction by excess substrate. Data indicated that the inhibition arises from lactate binding at the active site when flavin is under the Flsq oxidation-reduction state. The resulting ligand prevents transfer of the Flsq electron to heme b2 and ferricyanide. Y143F mutant enzyme is inhibited at high salt concentrations in contrast to the wild-type enzyme.

Analysis of the enzymatic properties of the D282N mutant of flavocytochrome b(sub 2) indicates that the pK(sub a) of His373 decreases and the transition state for proton removal destabilizes due to the mutation. The energy of the transition state for proton removal increases by 2.8 kcal/mol. Steady-state inhibition studies with the active-site ligands; sulfite, D-lactate, oxalate and pyruvate; indicate that binding for oxalate is maximally influenced. Analysis of the intermolecular hydrogen transfer reaction reveals that the mutation helps in the exchange of the histidine-bound proton.

Site-directed mutagenic studies on the role of phenolic tyrosine 143 in the flavocytochrome b2-catalyzed lactate dehydrogenation reaction reveal that Tyr143 forms a hydrogen bond with the Michaelis complex's carboxylate, resulting in stabilization of the complex. Tyr-143 does not cause electrophilic catalysis as hydrogen bond is weak in the transition state. The mutagenic studies compare reaction rates and kinetic isotopic effects between wild-type flavocytochromic b2 and its Y143F mutant.

A number of heme domain side chain substitutions in and around the interdomain interface for binding to its antigen is used to probe their effect on flavin and heme electron transfer. Several hydrophobic interactions and van der Waals contacts that are important for a catalytically competent docking of the heme domain onto the FDH domain and an isosbestic wavelength between oxidized and reduced heme for specifically following the kinetics of flavosemiquinone formation from two-electron reduced flavin is also discussed.

A study explains the reactivity of the flavin of flavocytochrome [b.sub.2], a flavohemoprotein with molecular oxygen. The described process of electron transfer is found to be extremely slow and incompetent.

The fully analytical treatment of the kinetics of reversible electron transfer (ET) in a catalytic system in the case of a minimal redox chain, consisting of a single one-electron relay connecting the electrode to a two-electron active site is provided. The results show that when intramolecular ET is not much faster than turnover, the active site can have apparent redox properties under turnover conditions, which differ from those, determined at equilibrium.

The crystal structure of the rat kidney long chain L-2-hydroxy acid oxidase (LCHAO) isozyme beta-1 and kinetic experiments designed to test the enzyme activity toward long chain alpha-hydroxy acids is described. A comparison of models of the substrate suggests that the very different reactivity with mandelate of LCHAO, spinach glycolate oxidase (GOX), yeast flavocytochrome b2 (FCB2), and mandelate dehydrogenase (MDH) could not be a hydride transfer mechanism.

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2AT. The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75 °C, respectively, and half lives of 9 and 5 h at 80 and 90 °C, respectively. The sequence of the 25 NH2-terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products. Article Highlights: The purification of subtilisin (SAPN) from M. thermohalophilus Nari2A was carried out. The molecular weight and the NH2-terminal sequence of the subtilisin were determined. Optimum pH and temperature values for activity were pH 10 and 75 °C respectively. SAPN was found to be effective in the deproteinization of crab and shrimp by-products. SAPN may be used as candidate for chitin extraction from crustacean by-products. [ABSTRACT FROM AUTHOR]

Thymotaxin, a Chemotactic Protein, Is Identical to [β.sub.2]-Microglobulin CHEMOTAXIS IS THE DIRECTIONAL migration of cells along soluble gradients of chemical substances. In mammals, several cell types are able to migrate [...]