11 research outputs found
The kinetic mechanism of beef kidney D-aspartate oxidase
The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate
FLUORESCENCE QUENCHING FOR FLAVINS INTERACTING WITH EGG WHITE RIBOFLAVIN-BINDING PROTEIN
Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum
The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s−1 and 105 s−1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s−1 for BCO and 232 s−1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Δ5-6→Δ4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s−1). With oxidized SCO and BCO the rate of isomerization is much faster (≈ 300 s−1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates ≈ 250 s−1 and 1.3 s−1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme product complexes
Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum
Health Care Administrators' Cardiac Rehabilitation Attitudes (HACRA) in North and South America and the Development of a Scale to Assess Them
A deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee
The honeybee genome has substantially fewer protein coding genes (approximate to 11 000 genes) than Drosophila melanogaster (approximate to 13 500) and Anopheles gambiae (approximate to 14 000). Some of the most marked differences occur in three superfamilies encoding xenobiotic detoxifying enzymes. Specifically there are only about half as many glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) in the honeybee. This includes 10-fold or greater shortfalls in the numbers of Delta and Epsilon GSTs and CYP4 P450s, members of which clades have been recurrently associated with insecticide resistance in other species. These shortfalls may contribute to the sensitivity of the honeybee to insecticides. On the other hand there are some recent radiations in CYP6, CYP9 and certain CCE clades in A. mellifera that could be associated with the evolution of the hormonal and chemosensory processes underpinning its highly organized eusociality