Pyruvic acid oxidase

Figure 8. Migration of the side chain during hydroxylation of phenylpyruvates with (hydroxy) phenyl-pyruvic acid oxidase...

The other pyruvic acid oxidases which have recently been described are additional examples which illustrate these same principles. They differ somewhat from each other either in the method of handling the hydrogens or in the ultimate fate of the 2-carbon residue. In Clostridium hutylicum, the end products are acetyl phosphate, CO2, and H2. In these reactions, the hydrogen removed from pyruvate is not used to reduce oxygen to the state of water but instead is liberated as hydrogen gas. Similarly, in the pyruvate dismutation reaction, extensively studied by Korkes and his co-workers, acetyl-CoA or acetyl phosphate represents the fate of the C2 fragment,... 

Pyruvic acid Enzyme electrode Fixed pyruvic acid oxidase Diagnosis Oxygen electrode... 

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

Experimental support for the mechanism of Eq. 15-26 has been obtained using D-chloroalanine as a substrate for D-amino acid oxidase.252-254 Chloro-pyruvate is the expected product, but under anaerobic conditions pyruvate was formed. Kinetic data obtained with a-2H and a-3H substrates suggested a common intermediate for formation of both pyruvate and chloro-pyruvate. This intermediate could be an anion formed by loss of H+ either from alanine or from a C-4a adduct. The anion could eliminate chloride ion as indicated by the dashed arrows in the following structure. This would lead to formation of pyruvate without reduction of the flavin. Alternatively, the electrons from the carbanion could flow into the flavin (green arrows), reducing it as in Eq. 15-26. A similar mechanism has been suggested for other flavoenzymes 249/255 Objections to the carbanion mechanism are the expected... 

Inaba et al. [2] d-Alanine Fish sauces D-Amino acid oxidase (d-AAOx) and pyruvate oxidase (PyOx)/PyOx is immobilised on a membrane and sandwiched between dialysis membrane and Teflon membrane of the oxygen electrode Oxygen electrode ... 

Y. Inaba, K. Mizukami, N. Hamada-Sato, T. Kobayashi, C. Imada and E. Watanabe, Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of d-amino acid oxidase and pyruvate oxidase, Biosens. Bioelectron., 19(5) (2003) 423-431. 

EC 1.11.1.7) (68) and diphenol oxidase (EC 1.10.3.1) (69) have been identified. The potential role of pyruvic decarboxylase (EC 4.1.1.1) catalyzed reaction as a source of acetaldehyde and other aldehydes in juice was discussed (70). Raymond et al. (71) isolated the decarboxylase from orange juice sections and demonstrated that only 10 to 15% of the enzyme was in an active form. Since the purified enzyme was only active with pyruvic acid and 2-ketobutyric acid of the series of 2-ketoacids examined, they (71) concluded that the direct contribution of orange pyruvic decarboxylase to the orange volatile profile was limited to acetaldehyde and possibly propionaldehyde. 

The crude enzyme preparation was found to catalyse the conversion of cadaverine (16) mainly into 17-oxosparteine (27) in the presence of pyruvic acid. The pyruvic acid served as a receptor for the amino-groups of (16) in a transamination reaction, having manifestly a close relationship to alkaloid formation.11 Diamine oxidase activity might have been expected to account for the... 

Berberine inhibits oxidative decarboxylation of yeast pyruvic acid (310) the same dose has, however, no effect upon aerobic glycolysis, Warburg s respiratory enzymes, indophenol oxidase, etc. Berberine and tetrahydroberberine have an inhibitory effect on oxidation of (+ )-alanine in rat kidney homogenates (498). Berberine and palmatine show a specific inhibitory effect upon cholinesterase in rabbit spleen and on pseudocholinesterase in horse serum (499). Berberine inhibits cellular respiration in ascitic tumors and even in tissue cultures (500-502). The specific toxic effect of berberine on the respiration of cells of ascitic tumors in mice was described (310). The glycolysis was not found to be affected, but the uptake of oxygen was smaller. Fluorescence was used in order to demonstrate berberine in cellular granules. Hirsch (503) assumed that respiration is inhibited by the effect of berberine on the yellow respiratory enzymes. Since the tumorous tissue contains a smaller number of yellow respiratory enzymes than normal tissue it is more readily affected by berberine. Subcutaneous injections of berberine, palmatine, or tetrahydropalmatine significantly reduce the content of ascorbic acid in the suprarenals, which is not affected by hypophysectomy (504). 

Organic acids are responsible for the acidity of honey and contribute considerably to its unique flavor (Anklam, 1998). Organic acid content of about 0.57% consists primarily of gluconic acid. It is a by-product of the enzymatic action of glucose oxidase on glucose (Olaitan et ah, 2007). Other organic acids identified up to the present are the pyruvic acid, malic acid, citric acid, succinic acid, and fumaric acid. 

The RebD enzyme was characterized as the first member of a new subfamily of heme-containing oxidases [34,36]. The enzyme acted as both a catalase and a CPA synthase, apparently converting two molecules of 7-chloroindole-3-pyruvic acid imine into ll,ll -dichloro-CPA. Formation of CPA by StaD, an enzyme homolog to RebD, was also confirmed for STA biosynthesis [35],... 

An interesting application for the oxidation of organic compounds is of electrochemical nature. Octacyano complexes have been used to monitor redox enzymes such as lactate oxidase (from Pediococcus sp.) and sarcosine oxidase (from Arthrobactersp.) in a suitable electrochemical system (114). Two equivalents of [M(CN)g] can, for example, be oxidized at the electrode surface to [M(CN)g], which in turn can oxidize the flavoproteien to its oxidized form. This in turn reacts with, for example, L-lactic acid to produce pyruvic acid. 

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. 

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). 

To understand the carbanion mechanism in flavocytochrome 62 it is useful to first consider work carried out on related flavoenzymes. An investigation into o-amino acid oxidase by Walsh et al. 107), revealed that pyruvate was produced as a by-product of the oxidation of )8-chloroalanine to chloropyruvate. This observation was interpreted as evidence for a mechanism in which the initial step was C -H abstraction to form a carbanion intermediate. This intermediate would then be oxidized to form chloropyruvate or would undergo halogen elimination to form an enamine with subsequent ketonization to yield pyruvate. The analogous reaction of lactate oxidase with jS-chlorolactate gave similar results 108) and it was proposed that these flavoenzymes worked by a common mechanism. Further evidence consistent with these proposals was obtained by inactivation studies of flavin oxidases with acetylenic substrates, wherein the carbanion intermediate can lead to an allenic carbanion, which can then form a stable covalent adduct with the flavin group 109). Finally, it was noted that preformed nitroalkane carbanions, such as ethane nitronate, acted as substrates of D-amino acid oxidase 110). Thus three lines of experimental evidence were consistent with a carbanion mechanism in flavoenzymes such as D-amino acid oxidase. 

The simplest example of such reactions is the decarboxylation of pyruvate. Both model and enzyme studies have shown the intermediacy of covalent complexes formed between the cofactor and the substrate. Kluger and coworkers have studied extensively the chemical and enzymatic behavior of the pyruvate and acetaldehyde complexes of ThDP (2-lactyl or LThDP, and 2-hydroxyethylThDP or HEThDP, respectively) . As Scheme 1 indicates, the coenzyme catalyzes both nonoxidative and oxidative pathways of pyruvate decarboxylation. The latter reactions are of immense consequence in human physiology. While the oxidation is a complex process, requiring an oxidizing agent (lipoic acid in the a-keto acid dehydrogenases , or flavin adenine dinucleotide, FAD or nicotinamide adenine dinucleotide , NAD " in the a-keto acid oxidases and Fe4.S4 in the pyruvate-ferredoxin oxidoreductase ) in addition to ThDP, it is generally accepted that the enamine is the substrate for the oxidation reactions. 

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

Tryptophan 331 is converted to tryptamine 332 by both aromatic L-amino acid decarboxylase (EC 4.1.1.28) and tyrosine decarboxylase (EC 4.1.1.25), and in both instances (334, 335) it was shown, either by use of the pro-R specific monoamine oxidase (335) or by degradation of the labeled tryptamines to glycine and use of the pro-S specific D-amino acid oxidase and pro-R specific glutamate pyruvate transaminase (334), that decarboxylation involved retention of configuration. Hydroxylation that leads to sporidesmin 333 has been shown to involve specific loss of the 3-pro-R hydrogen, and so again hydroxylation involves retention of configuration (102). 

Evidence (104,116) suggests that D-tryptophan is not fully utilized by human subjects and may have harmful effects. Further studies showed that D-tryptophan did not maintain nitrogen balance in normal young men (106), and in another study (117), urine from normal human subjects, after ingestion of D-tryptophan, contained a considerable portion of this compound as well as D-kynurenine. In contrast, the rat utilizes D-tryptophan completely however, food intake is significantly less in D-tryptophan-fed rats than in rats fed a diet containing L-tryptophan (110). The metabolic conversion of the D- to the L-enantiomer takes place in the rat liver and kidney D-amino acid oxidase plays a key role in this conversion (109). Indole pyruvic acid can be converted to L-tryptophan by a stereospecific transaminase apparently absent in humans The chick, on the other hand, utilizes only 7-40% of the D-tryptophan (82,83,110, 113). This wide range of values is probably due to different experimental conditions. D-tryptophan and... 

Some special methods of enantioselective electrochemical reactions should be mentioned. D-Alanine was prepared with an ee close to 100% using the electrochemical reduction of pyruvic acid using an electrode on which amino acid oxidase and electron mediator were immobilized... 

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