Pyruvate oxidase

A tri-enzymatic sensing layer based on kinase-oxidase activities for the detection of acetate was also described. A reaction sequence using acetate kinase, pyruvate kinase and pyruvate oxidase enabled the production of H2O2 in response to acetate injection in the range 10 pM - 100 mM59. 

A FIA system has been proposed for the CL detection of phosphate based on an enzymatic reaction and the application of a subsequent luminol reaction [41], The system consists of an immobilized pyruvate oxidase column, a mixing chamber for the CL reaction, and a PMT. H202 is generated by the reaction of phosphate and pyruvate oxidase and then reacts with luminol and HRP, producing... 

The pyruvate oxidase system (p. 139) should now be interpreted as pyruvate dihydrogenase together with the enzymes of the tricarboxylic acid cycle. The fact that sodium fluoroacetate itself did not poison the tricarboxylic acid cycle enzymes in... 

PHOSPHATE ACETYLTRANSFERASE PYRUVATE OXIDASE O-Acetylserine sulfhydralase,... 

NITRATE REDUCTASE NITRITE REDUCTASE PHENOL HYDROXYLASE PROLINE DEHYDROGENASE PUTRESCINE OXIDASE PYRUVATE OXIDASE SALICYLATE 1-MONOOXYGENASE SUCCINATE DEHYDROGENASE SULFITE REDUCTASE XANTHINE OXIDASE Falling ball viscometry,... 

HORSERADISH PEROXIDASE LACTOPEROXIDASE LIGNAN PEROXIDASE LYSYL OXIDASE MANGANESE PEROXIDASE MYELOPEROXIDASE OVOPEROXIDASE PEROXIDASE PYRUVATE OXIDASE XANTHINE OXIDASE Hydrogen selenide,... 

PROTOPORPHYRINOGEN OXIDASE PUTRESCINE OXIDASE PYRUVATE OXIDASE QUERCETIN 2,3-DIOXYGENASE... 

PYRUVATE, ORTHOPHOSPHATE DIKINASE PYRUVATE OXIDASE Pyruvate, phosphate dikinase,... 

Figure 6.21. Diedrich s model of pyruvate oxidase system.

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). 

By 1998, X-ray structures had been determined for four thiamin diphosphate-dependent enzymes (1) a bacterial pyruvate oxidase,119120 (2) yeast and bacterial pyruvate decarboxylases,121 122c (3) transketolase,110123124 and (4) benzoylformate decarboxylase.1243 Tire reactions catalyzed by these enzymes are all quite different, as are the sequences of the proteins. However, the thiamin diphosphate is bound in a similar way in all of them. 

Figure 14-2 (A) Stereoscopic view of the active site of pyruvate oxidase from the bacterium Lactobacillus plantarium showing the thiamin diphosphate as well as the flavin part of the bound FAD. The planar structure of the part of the intermediate enamine that arises from pyruvate is shown by dotted lines. Only some residues that may be important for catalysis are displayed G35 , S36 , E59 , H89 , F12T, Q122 , R264, F479, and E483. Courtesy of Georg E. Schulz.119 (B) Simplified view with some atoms labeled and some side chains omitted. The atoms of the hypothetical enamine that are formed from pyruvate, by decarboxylation, are shown in green.

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. 

Pyruvate oxidase. The soluble flavoprotein pyruvate oxidase, which was discussed briefly in Chapter 14 (Fig. 14-2, Eq. 14-22), acts together with a membrane-bound electron transport system to convert pyruvate to acetyl phosphate and C02.319 Thiamin diphosphate is needed by this enzyme but lipoic acid is not. The flavin probably dehydrogenates the thiamin-bound intermediate to 2-acetylthiamin as shown in Eq. 15-34. The electron acceptor is the bound FAD and the reaction may occur in two steps as shown with a thiamin diphosphate radical intermediate.3193 Reaction with inorganic phosphate generates the energy storage metabolite acetyl phosphate. 

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. 

Hydrophobic species bearing hydrocarbon chains present vitamin B12 or vitamin B6 type activity [5.37]. Such systems lend themselves to inclusion in membrane or micellar media. They thus provide a link with catalysis in more or less organized media such as membranes, vesicles, micelles, polymers [5.39-5.41] (see Section 7.4). Water soluble cyclophanes showing, for example, transaminase [5.42], acetyl transfer [5.43], pyruvate oxidase [5.44] or nucleophilic substitution [5.45] activity have been described. 

The -SH groups of dimercaptopropanol react with heavy metal ions including arsenic, to form very stable five-membered chelate rings, displacing heavy metal ions that would otherwise bind to essential -SH groups of enzymes such as succinoxidase and pyruvic oxidase. In this way, most of the enzyme activity can be restored if therapy is commenced soon after exposure. 

Coupling of a second enzyme reaction NAD(P)H+02->NADH oxidase NAD(P)++H20a->H202 monitoring [26] (For the particular case of lactate dehydrogenase, where pyruvate is one of the reaction products, pyruvate oxidase or pyruvate transaminase has been coupled to the main enzyme reaction) [23,27]... 

Other types of enzymes When no oxidase or dehydrogenase is available for a target analyte, other types of enzymes have been used for biospedfic recognition e.g. for citric acid detection, citrate lyase, and amperometric detection was possible by coupling to two more enzymatic reactions oxaloacetate decarboxylase and pyruvate oxidase, which convert citric add into H2O2 with the latter being monitored amperometrically with an H202 probe. For detection of acetic add, acetate kinase is used, coupled to pyruvate kinase and pyruvate oxidase [34,35]. 

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