Oxidation of Lactate

Another distinctive property of Acetobacter is the ability to oxidize d- and L-lactate to CO2 and H2O. This test utilizes a medium containing calcium lactate as both a carbon source and indicator. Growth of Acetobacter on lactate and subsequent over-oxidation yielding GO2 and H2O result in formation of a calcium carbonate precipitate. Gluconobacter grows poorly on this medium and does not oxidize enough of the lactate to raise the pH to the point at which GaCOj forms. 

Mix and dissolve the following ingredients. Yeast extract Calcium lactate Distilled water Agar 

Once dissolved, dilute to volume (lOOOmL) with distilled water. Adjust to pH 4.5 using 50% v/v HsP04 or 6M KOH. 

After autoclaving at 121 C/250°F for 15 min, place the container with the medium into a 45 C/H3°F to 50°G/122°F water bath. Once the temperature equilibrates, aseptically add high prooP ethanol or 

Mix thoroughly and prepare pour plates (25mL medium per plate). 

---

Biochemical oxidation of lactate to pymvate by lactate dehydrogenase is a well-known enzymatic reaction ia metaboHc pathways. 

The oxidation of lactate ion by alkaline permanganate (0.1 M base) was examined as part of the general study of the oxidation of acetone and its possible oxidation products . The reaetion is first-order in lactate ion, and, if it is assumed that it is also first-order in oxidant, then the second-order rate coefficient is 2 l.mole sec" at 25 °C. 

A spore-forming strain of Desulfitobacterium chlororespirans was able to couple the dechlorination of 3-chloro-4-hydroxybenzoate to the oxidation of lactate to acetate, pyruvate, or formate (Sanford et al. 1996). Whereas 2,4,6-trichlorophenol and 2,4,6-tribro-mophenol supported growth with the production of 4-chlorophenol and 4-bromophenol, neither 2-bromophenol nor 2-iodophenol was able to do so. The membrane-bound dehalogenase contains cobalamin, iron, and acid-labile sulfur, and is apparently specific for ortho-substituted phenols (Krasotkina et al. 2001). 

Sanford RA, JR Cole, EE Loffler, JM Tiedje (1996) Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroxybenzoate. Appl Environ Microbiol 62 3800-3808. 

Grootveld et al. (1994) employed this technique to investigate radiolytic, damage to biomolecules present in human body fluids. 7-Radiolysis of healthy or rheumatoid human serum (5.00 kGy) in the presence of atmospheric O2 gave rise to reproducible elevations in the concentration of NMR-detectable acetate, which are predominantly ascribable to the prior oxidation of lactate to pyruvate by OH radical followed by oxidative decarboxylation of pyruvate by radiolytically generated H2O2 and/or further OH radicals (Equations 1.7 and 1.8). 

Simon et al. [92] investigated a biocatalytic anode based on lactate oxidation by lactate dehydrogenase (LDH). The anodic current is generated by the oxidation of NADH (produced by NAD+ and substrate) while LDH catalyzes the electro-oxidation of lactate into pyruvate. As previously mentioned, the oxidation of NADH at bare electrodes requires a large overpotential, so these authors used poly(aniline) films doped with polyanions to catalyze NADH oxidation. Subsequent research by this group focused on targeting mutants of LDH that are amenable to immobilization on the polyaniline surface [93],... 

Note that oxidation of lactate utilises a proton so that, essentially, lactic acid is oxidised. 

Kinetic studies of hexacyanoferrate(III) oxidations have included the much-studied reaction with iodide and oxidation of the TICI2 anion, of hydrazine and hydrazinium, and of phenylhydrazine and 4-bromophenylhydrazine. These last reactions proceed by outer-sphere mechanisms, and conform to Marcus s theory. Catalyzed [Fe(CN)g] oxidations have included chlororuthenium-catalyzed oxidation of cyclohexanol, ruthenium(III)-catalyzed oxidation of 2-aminoethanol and of 3-aminopropanol, ruthenium(VI)-catalyzed oxidation of lactate, tartrate, and glycolate, and osmium(VIII)-catalyzed oxidation of benzyl alcohol and benzylamine. ... 

The active form of lactate dehydrogenase (mass 144 kDa) is a tetramer consisting of four subunits (1). Each monomer is formed by a peptide chain of 334 amino acids (36 kDa). In the tetramer, the subunits occupy equivalent positions (1) each monomer has an active center. Depending on metabolic conditions, LDH catalyzes NADH-de-pendent reduction of pyruvate to lactate, or NAD -dependent oxidation of lactate to pyruvate (see p. 18). 

The equilibrium of the reaction strongly favors lactate formation. At high concentrations of lactate and NAD"", however, oxidation of lactate to pyruvate is also possible (see p. 18). LDH catalyzes the reaction in both directions, but—like all enzymes—it has no effect on chemical equilibrium. 

Exactly the same steps occur during the oxidation of lactate to pyruvate, but in the opposite direction. As mentioned earlier, the direction which the reaction takes depends not on the enzyme, but on the equilibrium state—i.e., on the concentrations of all the reactants and the pH value (see p. 18). 

Cyclic voltammograms of the COx/Cyt.c electrode corresponding to the bioelectrocatalyzed reduction of 02 (i), and to the reference system, where 02 is excluded (ii). (c) Assembly of an integrated LDH electrode for the bioelectrocatalyzed oxidation of lactate by the surface cross-linking of an affinity complex formed between LDH and different structures of a boronate-linked PQQ-NAD monolayer. Parts (a) and (b) Reproduced from Ref. 27 by permission of the Royal Society of Chemistry (RSC). Part (c) Reproduced with permission from Ref. 25. Copyright 2002 American Chemical Society. 

RGURE 7 An oxidation-reduction reaction. Shown here is the oxidation of lactate to pyruvate. In this dehydrogenation, two electrons and two hydrogen ions (the equivalent of two hydrogen atoms) are removed from C-2 of lactate, an alcohol, to form pyruvate, a ketone. In cells the reaction is catalyzed by lactate dehydrogenase and the electrons are transferred to a cofactor called nicotinamide adenine dinucleotide. This reaction is fully reversible pyruvate can be reduced by electrons from the cofactor. In Chapter 13 we discuss the factors that determine the direction of a reaction. 

The different LDH isozymes have significantly different values of Vmax and KM, particularly for pyruvate. The properties of LDH4 favor rapid reduction of very low concentrations of pyruvate to lactate in skeletal muscle, whereas those of isozyme LDHx favor rapid oxidation of lactate to pyruvate in the heart. 

Propionibacteria can produce pyruvate either from glucose, primarily via the EMP pathway, or from oxidation of lactate by using a flavo-protein as a hydrogen acceptor (Gottschalk 1979). As shown in Figure... 

The NAD-pyruvate is formed by the enzyme from NAD+ and the enol form of pyruvate54 (the keto tautomer is the substrate for the normal reaction). The mechanism for the reaction is presumably catalyzed by His-195 in a manner similar to that of the oxidation of lactate (mechanism 16.9). 

Flavocytochrome b2 catalyses the oxidation of lactate to pyruvate at the expense of cytochrome C. After reduction of flavin (FMN) by the substrate, reducing equivalents are transferred to heme b2 and from there to cytochrome C602. The mechanism of this process has been studied603 at 5.0 °C by determining the D KIE in the FMN reduction using L-[2-2H]lactate and wild-type enzyme and also with the Y143F mutant prepared from transformed Escherichia coli604. Tritium IE in the conversion of [2-3-H]lactate to... 

Gemelli, L., Martino, G. and Tota, B. (1980). Oxidation of lactate in the compact and spongy myocardium of tuna fish. Comparative Biochemistry and Physiology 65B, 321-326. 

However, benzyl alcohols can be converted to benzaldehydes even in the dark.208 It has been reported that catalytic amounts of HBr may be used for this reaction.209 Primary aliphatic alcohols give rise to carboxylic esters. Catalytic amounts of bromine itself have been used, instead of HBr in the oxidation of lactate esters to pyruvates.210... 

Flavocytochrome b2 from Saccharomyces cerevisiae, a member of the FMN-dependent oxidoreductase superfamily, catalyzes the two-electron oxidation of lactate to pyruvate with subsequent electron-transfer to cytochrome c via the bound flavin [55], What distinguishes the enzyme from other family members is the N-terminal fusion of a heme-binding domain to the ySa-barrel structure, which hosts the primary active site. Rather than dumping the electrons from the reduced flavin hydroquinone onto molecular oxygen, they are transferred intramolecularly to the heme-binding domain and from there in a second intermolecular step to cytochrome c. 

Oxidation of lactate to pyruvate is inhibited, leading to a reduction in gluconeogenesis and accumulation of lactate (pyruvate + NADH + H — lactate + NAD ). 

Assay of lactate dehydrogenase illustrates this technique. This enzyme catalyzes the oxidation of lactate, yielding pyruvate and NADH as shown in Figure 6-17. NADH produced in this reaction can be used along with the intermediate electron carrier, phenazine methosulfate (PMS), to... 

Figure 6-17. Reactions involved in coupling oxidation of lactate with the reduction of p-nitroblue tetrazolium chloride.

Note that ethanol consumption leads to an accumulation ofNADH. This high concentration of NADH inhibits gluconeogenesis by preventing the oxidation of lactate to pyruvate. In fact, the high concentration ofNADH will cause the reverse reaction to predominate, and lactate will accumulate. The consequences may be hypoglycemia and lactic acidosis. 

Figure 26. Coupling of the photoswitchable interactions between Cyt c and a (20a)-pyridine mixed monolayer with (A) the reduction of O2 by COx and (B) the oxidation of lactate by LDH. (C) When the electrode is in the cationic merocyanine state (20b), repulsive interactions disallow the functioning of electrocatalytic processes.

The fermentation involves complete oxidation of lactate to CO2, generating reducing equivalents, which reduce sulfate to H2S ... 

LDH is a cytoplasmic enzyme which catalyzes the oxidation of lactate into pyruvate. It consists of 5 isoenzymes. As a ubiquitous enzyme and constituent of almost all tissues, LDH enters the blood circulation even in minor cell and tissue damage. The isoenzymes LDH4 and LDHs are predominantly located in the liver, cardiac muscle and malignant tissues. After severe damage of the liver parenchyma, these isoenzymes may become elevated. The half-life of LDH5 is very short (10 2 hours). 

Oxidation of lactate after heavy muscle work seems to be another example of mitochondrial function dealing with removal of a harmful compound responsible for dangerous acidosis of the tissue. It was found that the ATP formation coupled to lactate... 

Scheme 11. Idealized sketch showing the electroen matic oxidation of L-lactate at gold modified electrode surfaces, (a) Lactate dehydrogenase bound to CB-terminated alkylthiol SAMs prepared by covalent attachment of CB to 3-mercaptopropionic acid SAM derivatized with 1,4-diaminobutane. The electroenzymatic oxidation of lactate is observed only in the presence of soluble coenzyme (NAD" ") and a redox mediator (phenazine methosulfate) [215]. (b) Lactate deh3tdrogenase bound to NAD-terminated alkylthiol SAMs prepared by covalent attachment of Af -(2-aminoethyl)-NAD to a cystamine SAM derivatized with pjrrroloquinoline quinone. The reconstituted enzyme is electrically wired to the electrode surface via two NAD" -binding pockets involved in the affinity-binding surface reaction [242].

Sodium dichloroacetate (DCA) is a small molecule that has multiple effects on intermediary metabolism. Of primary interest in the current example is the ability of DCA to activate pyruvate dehydrogenase, the rate-limiting enzyme for the conversion of pyruvate to acetyl CoA. The pyruvate concentration is, in turn, replenished by oxidation of lactate, thereby replenishing concentrations of the latter. Such a reduction may decrease the morbidity in head trauma, where local (CSF) elevated lactate is thought to be neurotoxic. 

Figure 3-23. The assembly of an integrated lactate dehydrogenase, LDH, electrode for the bioelectrocatalyzed oxidation of lactate by the crosslinking of an affinity complex between LDH and PQQ on a polyacryhc acid polyaniline lihn composite associated with the electrode. Reproduced with permission from ref. 34. Copyright 2002 American Chemical Society.

---