Lactate dehydrogenation

Deuterium and tritium isotope effects in the lactate dehydrogenations by fiavocy-tochrome b2... 

As a respiratory enzyme, the production of flavocytochrome bi is induced by the presence of oxygen and, more specifically, L-lactate. In addition to providing pyruvate (the product of lactate oxidation) for the Krebs cycle, flavocytochrome bj also participates in a shorter respiratory chain that ultimately directs the energy gained from L-lactate dehydrogenation... 

FIGURE 4. Mechanisms for lactate dehydrogenation, a. By hydride transfer His373 abstracts the hydroxyl proton and promotes hydride transfer to the N-5 of the flavin, b. By carbanion formation His373 removes the OG-carbon hydrogen as a proton forming a carbanion. A subsequent transfer of two-electrons to the flavin is required. 

The interaction between Tyr 143 and the carboxylate of pyruvate in subunit 2 has already been mentiond (Section III,D). In subunit 1, however, where pyruvate is absent, Tyr 143 is now hydrogen bonded to an oxygen of one of the heme propionate groups (25). Thus it would appear that this residue is involved in both lactate dehydrogenation and interdomain communication. The possible central role of Tyr 143 will be discussed further in Section VI,D,3. 

Substitution of Ala 306, a residue in the proteinase-sensitive loop of S. cerevisiae flavocytochrome 62 > by Ser results in small but significant changes in kcat and Km for L-lactate (143) (Table VIII). Thus, a rather minor alteration to a surface residue quite remote from the active site has an effect on lactate dehydrogenation. The result is similar to that obtained by proteolytic cleavage of the region in which the substitution... 

The measurement of lactate dehydrogenate (LDH EC. 1.1.1.27) in culture supernatants gives a quantitative value for the loss of cell viability ... 

We prepared pent- and hex-2-ulosonates according to Scheme 4 (61). The apparent values of the substrates are mostly < 1 mM (Table 16). They were determined with crude extract of P. mirabilis to which the substrates were added as salts at pH 8.5. The apparent Vmax values are 2-13 % of that for (/ )-lactate dehydrogenation (47,62). The absolute values are still rather high when compared with redox enzyme activities of many other microorganisms for rather simple substrates. Two examples with P. mirabilis cells grown on racemic lactate and dimethyl-sulphoxide will be briefly described. 

Daff and co-workers have investigated the catalytic cycle which can be described in terms of five consecutive electron-transfer events. Lactate dehydrogenation results in the two-electron reduction of FMN. The two electrons are individually passed to the 2-heme (intramolecular electron transfer) and then onto cytochrome c (intermolecular electron transfer). Freeze quench EPR was used to demonstrate that during steady-state turnover of the enzyme approximately 75% of the flavin is in the semiquinone state, and hence that this is a catalytic intermediate. 

Pyruvic acid is the simplest homologue of the a-keto acid, whose established procedures for synthesis are the dehydrative decarboxylation of tartaric acid and the hydrolysis of acetyl cyanide. On the other hand, vapor-phase contact oxidation of alkyl lactates to corresponding alkyl pyruvates using V2C - and MoOa-baseds mixed oxide catalysts has also been known [1-4]. Recently we found that pyruvic acid is obtained directly from a vapor-phase oxidative-dehydrogenation of lactic acid over iron phosphate catalysts with a P/Fe atomic ratio of 1.2 at a temperature around 230°C [5]. 

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. 

Dehydrogenases often act primarily to reduce a carbonyl compound rather than to dehydrogenate an alcohol. These enzymes may still be called dehydrogenases. For example, in the lactic acid fermentation lactate is formed by reduction of pyruvate but we still call the enzyme lactate dehydrogenase. In our bodies this enzyme functions in both directions. However, some enzymes that act mainly in the direction of reduction are called reductases. An example is aldose reductase, a member of a family of aldo-keto reductases71 73 which have (a / P)8-barrel structures.74 76... 

Some enzymes contain bound NAD+ which oxidizes a substrate alcohol to facilitate a reaction step and is then regenerated. For example, the malolactic enzyme found in some lactic acid bacteria and also in Ascaris decarboxylates L-malate to lactate (Eq. 15-12). This reaction is similar to those of isocitrate dehydrogenase,110-112 6-phosphogluconate dehydrogenase,113 and the malic enzyme (Eq. 13-45)114 which utilize free NAD+ to first dehydrogenate the substrate to a bound oxoacid whose (3 carbonyl group facilitates decarboxylation. Likewise, the bound NAD+ of the malolactic... 

The ratio [NAD+]/[NADH] appears to be maintained at a relatively constant value and in equilibrium with a series of different reduced and oxidized substrate pairs. Thus, it was observed that in the cytoplasm of rat liver cells, the dehydrogenations catalyzed by lactate dehydrogenase, sn-glycerol 3-phosphate dehydrogenase, and malate dehydrogenase are all at equilibrium with the same ratio of [NAD+]/[NADH].166 In one experiment rat livers were removed and frozen in less than 8 s by "freeze-clamping" (Section L,2) and the concentrations of different components of the cytoplasm determined167 the ratio [NAD+] / [NADH] was found to be 634, while the ratio of [lactate]/[pyruvate] was 14.2. From these values an... 

The NADH is derived from glyceraldehyde 3-phosphate dehydrogenation. Thus, yeast fermentation yields ethanol rather than lactate as an end product of glycolysis. Small amounts of ethanol are produced by the microbial flora of the gastrointestinal tract. Other types of fermentation using similar reactions occur in microorganisms and yield a variety of products (e.g., acetate, acetone, butanol, butyrate, isopropanol, hydrogen gas). 

The major reaction is oxidative dehydrogenation at the secondary hydroxyl site of lactic acid, but the product pyruvic acid in its free-acid form is unstable to decompose. Thus the substrate was supplied as ethyl ester to protect the carboxyl moiety. Esterification is also of benefit to vapor-phase flow operation in making acids more volatile. Hydrolysis of ethyl lactate gives free pyruvic acid with further decarboxylation to actaldehyde. Ethanol, which is another fragment of ester hydrolysis, eould be either oxidized to acetaldehyde or dehydrated to ethylene at higher temperature above 350°G. The reaction network is summerized in Scheme 1. 

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