Pyruvate oxidase oxidation

Chlorine, phosgene. Lewisite, and SM all react with thiol groups as well as produce oxidants. The arsenic group in Lewisite has a high affinity to the alpha and gamma thiol groups of lipoic acid found in enzymes (e.g., pyruvate oxidase). Oxidants occur as part of the normal metabolism of cells (redox homeostasis). In the diseased state (e.g., exposure to a chemical agent), there is an acute... 

Pyruvate oxidase Oxidative decarboxylation of pyruvic acid CH3COCOOH CH3COOH or to CH3COOPO3 + CO2 FAD... 

Pyruvic oxidase —oxidation of pyruvate to acetyl CoA, decarboxylation of pyruvate to acetoin, and dismutation of diacetyl to acetoin and acetate. 

Oxidative decarboxylations of a-keto acids are mediated by either enzymes having more than one cofactor or complex multienzyme systems utilizing a number of cofactors. For example, pyruvate oxidase uses TPP and FAD as coenzymes, the function of the latter being to oxidize the intermediate (41). Conversion of pyruvate to acetyl-CoA requires a multienzyme complex with the involvement of no less than five coenzymes, TPP, CoA, dihydrolipoate, FAD and NAD+ (74ACR40). 

A related reaction that is known to proceed through acetyl-TDP is the previously mentioned bacterial pyruvate oxidase. As seen in Fig. 14-2, this enzyme has its own oxidant, FAD, which is ready to accept the two electrons of Eq. 14-22 to produce bound acetyl-TDP. The electrons may be able to jump directly to the FAD, with thiamin and flavin radicals being formed at an intermediate stage.1353 The electron transfers as well as other aspects of oxidative decarboxylation are discussed in Chapter 15, Section C. 

Tire enzyme does not require lipoic acid. It seems likely that a thiamin-bound enamine is oxidized by an iron-sulfide center in the oxidoreductase to 2-acetyl-thiamin which then reacts with CoA. A free radical intermediate has been detected318 321 and the proposed sequence for oxidation of the enamine intermediate is that in Eq. 15-34 but with the Fe-S center as the electron acceptor. Like pyruvate oxidase, this enzyme transfers the acetyl group from acetylthiamin to coenzyme A. Cleavage of the resulting acetyl-CoA is used to generate ATR An indolepyruvate ferredoxin oxidoreductase has similar properties 322... 

Mechanism of thiamine pyrophosphate action. Intermediate (a) is represented as a resonance-stabilized species. It arises from the decarboxylation of the pyruvate-thiamine pyrophosphate addition compound shown at the left of (a) and in equation (2). It can react as a carbanion with acetaldehyde, pyruvate, or H+ to form (b), (c), or (d), depending on the specificity of the enzyme. It can also be oxidized to acetyl-thiamine pyrophosphate (TPP) (e) by other enzymes, such as pyruvate oxidase. The intermediates (b) through (e) are further transformed to the products shown by the actions of specific enzymes. 

Recently, Schloss et al (33) showed that IM and TP were able to quantitatively displace a radiolabelled SU herbicide from ALS, indicating competitive binding. Curiously, the SU ligand was also displaced by the quinone, Qo. It was proposed that SU, TP, and IM bind to ALS in a vestigial quinone binding site associated with the evolution of ALS from pyruvate oxidase. This enzyme is an FAD-protein that catalyzes the oxidation of pyruvate to acetate. 

That there is an enamine-like intermediate in all thiamin diphosphate-dependent enzymatic pathways has been suggested for many years, although the issue is still sometimes clouded by the observation that the C2a-protonated form, the C2a-hydro-xyethylThDP when added to oxidases such as pyruvate oxidase (POX) and the pyruvate dehydrogenase multienzyme complex (PDHc), will undergo oxidation. 

Mercury interferes with mitochondrial oxidation in the brain through mercaptide formation with thiol groups in pyruvate oxidase. Succinic dehydrogenase of the citric acid cycle is also inhibited. 

Sensors have also been constructed from some oxidases directly contacted to electrodes to give bioelectrocatalytic systems. These enzymes utilize molecular oxygen as the electron acceptor for the oxidation of their substrates. Enzymes such as catechol oxidase, amino acid oxidase, glucose oxidase, lactate oxidase, pyruvate oxidase, alcohol oxidase, xanthine oxidase and cholesterol oxidase catalyze the oxidation of their respective substrates with the concomitant reduction of O2 to H2O2 ... 

Similarly, the pyruvate (oxidase) dehydrogenase complex (PYOX) can be activated directly by electrogenerated methyl viologen radical cations (MV" ) as mediator. Thus, the naturally PYOX-catalyzed oxidative decarboxylation of pyruvic acid in the presence of coenzyme A (HSCoA) to give acetylcoenzyme A (acetyl-SCoA) (see section on oxidases) can be reversed. In this way, electroenzymatic reductive carboxylation of acetyl-SCoA is made possible (Fig. 15). 

Pyruvate oxidase (Pyox) is a FAD- and thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the reaction of pyruvate to give acetyl phosphate or vice versa (see Fig. 15). If used in the oxidative way, it can be activated and reactivated under nonaerobic conditions using ferrocene mediators. Kinetic parameters of the indirect electrochemical process using the enzyme incorporated into a biomimetic supported bilayer at a gold electrode have been reported [142]. Similarly, FAD-dependent amino oxidases may also be applied. 

The different cosubstrate specificities of the lactate-oxidizing enzymes offer the use of a great variety of electrochemical indicator reactions in membrane sensors. In enzyme electrodes based on LDH the biochemical reaction has been coupled to the electrode via NADH oxidation, either directly or by using mediators or additional enzymes (see Section 3.2.1). This leads to a shift of the unfavorable reaction equilibrium by partial trapping of the reduced cofactor. Such a shift has also been achieved by using pyruvate oxidase coimmobilized with LDH (Mizutani, 1982). 

In these reactions, the C2-atom of ThDP must be deprotonated to allo v this atom to attack the carbonyl carbon of the different substrates. In all ThDP-dependent enzymes this nucleophilic attack of the deprotonated C2-atom of the coenzyme on the substrates results in the formation of a covalent adduct at the C2-atom of the thiazolium ring of the cofactor (Ila and Ilb in Scheme 16.1). This reaction requires protonation of the carbonyl oxygen of the substrate and sterical orientation of the substituents. In the next step during catalysis either CO2, as in the case of decarboxylating enzymes, or an aldo sugar, as in the case of transketo-lase, is eliminated, accompanied by the formation of an a-carbanion/enamine intermediate (Ilia and Illb in Scheme 16.1). Dependent on the enzyme this intermediate reacts either by elimination of an aldehyde, such as in pyruvate decarboxylase, or with a second substrate, such as in transketolase and acetohydroxyacid synthase. In these reaction steps proton transfer reactions are involved. Furthermore, the a-carbanion/enamine intermediate (Ilia in Scheme 16.1) can be oxidized in enzymes containing a second cofactor, such as in the a-ketoacid dehydrogenases and pyruvate oxidases. In principal, this oxidation reaction corresponds to a hydride transfer reaction. 

In the next section, the mechanism of the C2-H deprotonation of ThDP in enzymes is considered, followed by a discussion of the proton transfer reactions during catalysis. Finally, the oxidation mechanism of the a-carbanion/enamine intermediate in pyruvate oxidase is discussed. 

The pyruvate oxidase from Lactobacillus plantarum catalyzes the oxidative decarboxylation of pyruvate and the formation of acetylphosphate, CO2 and H2O2 in the presence of oxygen and phosphate (Gotz and Sedewitz, 1990 Sedewitz et al., 1984a Sedewitz et al., 1984b). Each subunit of the homotetrameric enzyme binds... 

Figure 16.4. Absorption spectra of oxidized (solid line) and reduced (dotted line) pyruvate oxidase from Lactobacillus plantarum.

For investigating pyruvate oxidase the spectroscopic properties of the isoalloxa-zine system of FAD are an excellent probe for monitoring steps relevant to enzyme catalysis directly. In pyruvate oxidase from Lactobacillus plantarum the lowest n-n transition can be used to distinguish between oxidized and reduced FAD (Fig. 

Figure 16.5. Reduction of oxidized pyruvate oxidase by different concentrations of the substrate pyruvate under anaerobic conditions in 0.2 M potassium phosphate buffer, pH 6.0. Pyruvate concentration 2.5 mM (open diamond), 5 mM (open square), 20 mM (open circle), and 50 mM (open triangle).

NADH), lactate oxidase (oxidizes lactate to pyruvate), and catalase (figure... 

This compound is the product of oxidation of the enamine by any one of the following oxidizing agents on enzymes most commonly by the dithiolane ring of lipoic acid covalently amidated to a lysine side chain in the 2-oxoacid dehydrogenase multienzyme complexes less frequently by FAD in the pyruvate oxidases — these come in two flavors, forming acetate in Escherichia coli and forming acetylphosphate in L. plantarum, finally by NAD. ... 

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