Lactate dehydrogenases reduction

Enzyme catalyzed reductions of carbonyl groups are more often than not com pletely stereoselective Pyruvic acid for example is converted exclusively to (5) (+) lactic acid by the lactate dehydrogenase NADH system (Section 15 11) The enantiomer... 

FIGURE 19.30 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD consumed in the glyceraldehyde-3-P dehydrogenase reaction, (b) In oxygen-depleted muscle, NAD is regenerated in the lactate dehydrogenase reaction. 

Many dehydrogenase enzymes catalyze oxidation/reduction reactions with the aid of nicotinamide cofactors. The electrochemical oxidation of nicotinamide adeniiw dinucleotide, NADH, has been studied in depthThe direct oxidation of NADH has been used to determine concentration of ethanol i s-isv, i62) lactate 157,160,162,163) pyTuvate 1 ), glucose-6-phosphate lactate dehydrogenase 159,161) alanine The direct oxidation often entails such complications as electrode surface pretreatment, interferences due to electrode operation at very positive potentials, and electrode fouling due to adsorption. Subsequent reaction of the NADH with peroxidase allows quantitation via the well established Clark electrode. 

Adam, W., Lazarus, M., Saha-Moller, C.R. and Schreier, P. (1998) Quantitative transformation of racemic 2-hydroxy acids into (R)-2-hydroxy acids by enantioselective oxidation with glycolate oxidase and subsequent reduction of 2-keto acids with D-lactate dehydrogenase. Tetrahedron Asymmetry, 9 (2), 351-355. 

Using two types of specially synthesized rhodium-complexes (12a/12b), pyruvate is chemically hydrogenated to produce racemic lactate. Within the mixture, both a d- and L-specific lactate dehydrogenase (d-/l-LDH) are co-immobilized, which oxidize the lactate back to pyruvate while reducing NAD+ to NADH (Scheme 43.4). The reduced cofactor is then used by the producing enzyme (ADH from horse liver, HL-ADH), to reduce a ketone to an alcohol. Two examples have been examined. The first example is the reduction of cyclohexanone to cyclohexanol, which proceeded to 100% conversion after 8 days, resulting in total TONs (TTNs) of 1500 for the Rh-complexes 12 and 50 for NAD. The second example concerns the reduction of ( )-2-norbornanone to 72% endo-norbor-nanol (38% ee) and 28% exo-norbornanol (>99% ee), which was also completed in 8 days, and resulted in the same TTNs as for the first case. 

The successful synthetic application of this electroenzymatic system has first been shown for the in-situ electroenzymatic reduction of pyruvate to D-lactate using the NADH-dependent D-lactate dehydrogenase. Electrolysis at — 0.6 V vs a Ag/AgCl-reference electrode of 50 mL of a 0.1 M tris-HCL buffer of pH 7.5 containing pentamethylcyclopentadienyl-2,2 -bipyridinechloro-rhodium(III) (1 x 10 3 M), NAD+ (2 x 10 3 M), pyruvate (2 x 10 2 M), 1300 units D-lactate dehydrogenase (divided cell, carbon foil electrode) after 3 h resulted in the formation of D-lactate (1.4 x 10 2 M) with an enantiomeric excess of 93.5%. This means that the reaction occurred at a rate of 5 turnovers per hour with respect to the mediator with a 70% turnover of the starting material. The current efficiency was 67% [67],... 

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). 

Isotope effects have also been applied extensively to studies of NAD+/NADP+-linked dehydrogenases. We typically treat these enzymes as systems whose catalytic rates are limited by product release. Nonetheless, Palm clearly demonstrated a primary tritium kinetic isotope effect on lactate dehydrogenase catalysis, a finding that indicated that the hydride transfer step is rate-contributing. Plapp s laboratory later demonstrated that liver alcohol dehydrogenase has an intrinsic /ch//cd isotope effect of 5.2 with ethanol and an intrinsic /ch//cd isotope effect of 3-6-4.3 with benzyl alcohol. Moreover, Klin-man reported the following intrinsic isotope effects in the reduction of p-substituted benzaldehydes by yeast alcohol dehydrogenase kn/ko for p-Br-benzaldehyde = 3.5 kulki) for p-Cl-benzaldehyde = 3.3 kulk for p-H-benzaldehyde = 3.0 kulk for p-CHs-benzaldehyde = 5.4 and kn/ko for p-CHsO-benzaldehyde = 3.4. 

Exposure of rats for 6 hours/day, 5 days/ week for 4 weeks to 15 ppm caused corneal lesions in some of the rats along with reductions in total serum protein, lactate dehydrogenase, and serum albumin. Rats exposed to... 

Assays are frequently needed to detect marked and acute cytotoxicity that may confound the interpretation of cell-based efficacy assays. Neutral red uptake is one of the most commonly used cytotoxicity assays and is used in the regulatory phototoxicity assay on NT3 fibroblasts [13]. It has been show to be more sensitive than assays for mitochondrial reductive capacity such as the tetrazolium reductase assays, ATP depletion assays, or for cell permeabilization or mpture such as dye uptake or lactate dehydrogenase leakage. Lysosomes take up, protonate and trap neutral red when cellular ATP production is sufficient to maintain pH gradients. 

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. 

When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of the pyruvate and NADH produced in glycolysis, NAD+ is regenerated from NADH by the reduction of pyruvate to lactate. As mentioned earlier, some tissues and cell types (such as erythrocytes, which have no mitochondria and thus cannot oxidize pyruvate to C02) produce lactate from glucose even under aerobic conditions. The reduction of pyruvate is catalyzed by lactate dehydrogenase, which forms the l isomer of lactate at pH 7 ... 

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... 

Hydroxy and Amino Acids. Reduction of 2-oxoacids with NADH. catalyzed by lactate dehydrogenase, is an established method for the preparation of hontochiral ur-hydroxy acids of both S and R configurations. The enzyme is found in all higher organisms and can be easily isolated from a variety of mammalian and bacterial sources. 

This system has been successfully applied to the in-situ electroenzymatic reduction of pyruvate to D-lactate using the NADH-dependent D-lactate dehydrogenase or the reduction of 4-phenyl-2-butanone to (5)-4-phenyl-2-butanol using the NADH-dependent horse liver alcohol dehydrogenase (HLADH) with high enantioselectivity (Fig. 22.4) [65]. 

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