Flavoprotein oxidase reactions

This chapter is not comprehensive since it reflects the interest of the author in the kinetic and chemical mechanism of flavoprotein oxidase reactions. It is meant to complement the special interests of others who have written excellent reviews from their respective view points. The... 

The initial steady state velocity data from flavoprotein oxidase reactions, when plotted in double reciprocal form, almost invariably generate families of parallel and usually straight lines see Figures la and lb). 

The expected result was obtained since n-amino acid oxidase converted )8-chloroalanine to pyruvate under anaerobic conditions, to chloropyruvate at high O2 concentrations, and to mixtures of these at intermediate O2 concentrations. Under steady state conditions, the reaction behaved as if cleavage of the a C-H bond were the rate-limiting process in turnover, although stopped-flow spectrophotometric measurements showed that this interpretation can not be entirely correct in this case (53) or in the case of )8-choloro-a-aminobutyrate (54), a-yS-Elimination has now been observed in three flavoprotein oxidase reactions (54) and can be considered strong circumstantial evidence for a-proton removal from compounds which closely resemble the physiological substrates. 

Flavins are very versatile redox coenzymes. Flavopro-teins are dehydrogenases, oxidases, and oxygenases that catalyze a variety of reactions on an equal variety of substrate types. Since these classes of enzymes do not consist exclusively of flavoproteins, it is difficult to define catalytic specificity for flavins. Biological electron acceptors and donors in flavin-mediated reactions can be two-electron acceptors, such as NAD+ or NADP+, or a variety of one-electron acceptor systems, such as cytochromes (Fe2+/ Fe3+) and quinones, and molecular oxygen is an electron acceptor for flavoprotein oxidases as well as the source of oxygen for oxygenases. The only obviously common aspect of flavin-dependent reactions is that all are redox reactions. 

Similar surface problems are encountered with the flavoprotein oxidases. The classical glucose sensor is straightforward. The reaction may be divided in the following steps ... 

Table 7.3 Reoxidation of Reduced Flavins in Flavoprotein Oxidases The overall reaction is ...

Fortunately, the characteristic absorbance of certain stable and transient enzyme species and, in some instances, of products, together with the fact that the two half-reactions can be studied separately, permits informative rapid kinetic measurements of the overall and partial reactions of flavoprotein oxidases. Stopped-flow spectrophotometric methods (26) have been particularly useful (the irreversibility of the partial and overall reactions rules out relaxation methods) because the measured rate constants often correspond in part or whole to the reciprocals of the steady state coefficients. This is the major reason for using the formulation... 

Oxidative Half-Reaction. The free, fully reduced flavoprotein oxidases react with O2 at rates varying between about 2 sec (37)... 

In sharp contrast to the reductive half-reaction, where the free oxidized flavin is totally inert in the presence of physiological substrates, reduced model flavins are appreciably reactive (nonenzymatically) with O2 and other electron acceptors. However, the O2 reactivity of reduced flavin is complicated for two perhaps related reasons (61). First, the reaction is autocatalytic owing to the formation of 2F (from F and FH2) which in its anionic state is extremely reactive with Oo. Second, the superoxide radical is an important kinetic intermediate in O2 reduction (59). Neither of these features is observed with the reduced flavoprotein oxidases. 

DAAO is one of the most extensively studied flavoprotein oxidases. The homodimeric enzyme catalyzes the strictly stere-ospecihc oxidative deamination of neutral and hydrophobic D-amino acids to give a-keto acids and ammonia (Fig. 3a). In the reductive half-reaction the D-amino acid substrate is converted to the imino acid product via hydride transfer (21). During the oxidative half-reaction, the imino acid is released and hydrolyzed. Mammalian and yeast DAAO share the same catalytic mechanism, but they differ in kinetic mechanism, catalytic efficiency, substrate specificity, and protein stability. The dimeric structures of the mammalian enzymes show a head-to-head mode of monomer-monomer interaction, which is different from the head-to-tail mode of dimerization observed in Rhodotorula gracilis DAAO (20). Benzoate is a potent competitive inhibitor of mammalian DAAO. Binding of this ligand strengthens the apoenzyme-flavin interaction and increases the conformational stability of the porcine enzyme. 

The enzyme D-lactate dehydrogenase from Megasphaera elsdenii catalyzes the oxidation of D-lactate to pyruvate, with an electron-transferring flavoprotein serving as the ultimate oxidant. Its reaction is similar to the first step of the lactate oxidase reaction, but the two enzymes use enantiomeric substrates, leading to the proposal that the two enzymes utilize similar mechanisms but bind their substrates in opposite orientations (Ghisla et al., 1976). Incubation of o-lactate dehydrogenase with d-13 leads to enzyme inactivation with a partition ratio of 5 (Olson et al., 1979). A novel pink chromophore formed concomitantly... 

Until recently, the amino acid oxidase reaction has been studied only in the direction of ammonia and a-keto acid formation. In the presence of air the reaction proceeds to completion and is essentially irreversible because of the reoxidation of the reduced flavoprotein by molecular oxygen [reaction (5)]. Meister and his associates 11, 12) have provided a clear demonstration of the reversibility of the amino acid oxidase reaction with D-amino acid oxidase (from sheep kidney) and L-amino acid oxidase (from snake venom). When an amino acid, ammonia, and the a-keto acid analog of a second amino acid are incubated with either amino acid oxidase under anaerobic conditions, the formation of the second amino acid is observed ... 

The above sequence of reactions is expected to interfere with attempts to observe SERRS from catalytically active GO at an Ag electrode. A similar problem may arise with other flavoprotein oxidases. Additionally, the Ag20 layer formed at the electrode surface may inhibit electron transfer between these proteins and the Ag electrode. 

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