Reduction of Pyruvate to Lactate

Cells that lack mitochondria (e.g., erythrocytes) or contain mitochondria but under hypoxic conditions (e.g., vigorously and repeatedly contracting muscle) reduce pyruvate to lactate by lactate dehydrogenase (LDH), which uses NADH as a reductant. This reduction regenerates NAD+, which is required for continued oxidation of glyc-eraldehyde 3-phosphate. 

This reversible reaction is the final step of glycolysis and is catalyzed by (LDH)  

The overall reaction of glycolysis becomes Glucose + 2ADP + 2Pf  

LDH (M.W. 134,000) oceurs as five tetrameric isoenzymes composed of two different types of subunits. Subunits M (for muscle) and H (for heart) are encoded by loci in chromosomes 11 and 12, respectively. Two subunits used in the formation of a tetramer yield five combinations H4(LDH-1), H3M(LDH-2), H2M2(LDH-3), HM3(LDH-4), and M4(LDH-5). The tissue distributiont of LDH isoenzymes is variable. For example, LDH-1 and LDH-2 are the principal isoenzymes in heart, kidney, brain, and erythrocytes LDH-3 and LDH-4 predominate in endocrine glands (e.g., thyroid, adrenal, pancreas), lymph nodes, thymus, spleen, leukocytes, platelets, and nongravid uterine muscle and LDH-4 and LDH-5 preponderate in liver and skeletal muscle. In tissue injury or insult, the appropriate tissue isoenzymes appear in plasma (Chapter 8) thus, determination of LDH isoenzyme composition has diagnostic value. 

Serum LDH isoenzymes can be separated by electrophoresis on agarose gel or cellulose acetate membrane, usually at pH 8.6. After separation, their location is determined by incubation of the support medium in a 

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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). 

Figure 6-1. The steps of glycolysis. Feedback inhibition of glucose phosphorylation by hexokinase, inhibition of pyruvate kinase, and the main regulatory, rate-limiting step catalyzed by phosphofructoki-nase (PFK-I) are indicated, pyruvate formation and substrate-level phosphorylation are the main outcomes of these reactions. Regeneration of NAD occurs by reduction of pyruvate to lactate during anaerobic glycolysis.

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 ... 

Figure 17-7 Outline of the glycolysis pathway by which hexoses are broken down to pyruvate. The ten enzymes needed to convert D-glucose to pyruvate are numbered. The pathway from glycogen using glycogen phosphorylase is also included, as is the reduction of pyruvate to lactate (step 11). Steps 6a-7, which are involved in ATP synthesis via thioester and acyl phosphate intermediates, are emphasized. See also Figures 10-2 and 10-3, which contain some additional information.

The final form of metabolic regulation is effected by the use of iso-enzymes, which are multiple forms of an enzyme. For example, lactate dehydrogenase exists in five forms in a rat. They differ in primary structure and have different isoelectric points, but they all catalyse the reversible reduction of pyruvate to lactate. 

Regeneration of NAD+ by reduction of pyruvate to lactate. Because NAD+ is a necessary participant in the oxidation of glyceraldehyde-3-phosphate to glycerate-l,3-bisphosphate, glycolysis is possible only if there is a way by which NADH can be reoxidized. 

Lactate dehydrogenase (LDH, E.C. 1.1.1.27) catalyzes the reduction of pyruvate to lactate using NADH as a cofactor ... 

Fig. I. Reduction of pyruvate to lactate, (a) Re attack giving L-lactate ((.S )-lactate). X is CH3. (b) Si attack giving D-lactate ((R(-lactate). X is CH3. When X is H, Re attack (a) and Si attack (b) both give glycollate.

The activity of LDH can be measured as the reduction of pyruvate to lactate (Vassault, 1983). The reduction is coupled to the oxidation of NADH to NAD+, which is followed spectrophotometrically at 340 nm. The equilibrium is far on the side of NAD+ and lactate. Because NADH has a high absorbance at 340 nm compared with NAD+, the reaction is measured as the rate of decrease in absorbence at 340 nm. 

As indicated, the reaction is reversible, and the reaction equilibrium strongly favors the reduction of pyruvate to lactate (P L)— the reverse reaction ... 

There are a number of forms of human lactate dehydrogenase, that is, what are variously denoted as LDH , LDH2, LDH3, LDH4, and LDH5 (e.g., Voet and Voet, 1995, pp. 183, 164 Hoffman, 1999, pp. 386, 387). The fifth form of the enzyme, sometimes denoted as the M-type, for muscle (and liver), is presumably the most efficient for the conversion (or reduction) of pyruvate to lactate, or pyruvic acid to lactic acid. 

For example, when a heart attack occurs, a lack of blood supplied to the heart muscle causes some of the heart muscle cells to die. These cells release their contents, including their enzymes, into the bloodstream. Simple tests can be done to measure the amounts of certain enzymes in the blood. Such tests, called enzyme assays, are very precise and specific because they are based on the specificity of the enzyme-substrate complex. If you wish to test for the enzyme lactate dehydrogenase (LDH), you need only to add the appropriate substrate, in this case pyruvate and NADH. The reaction that occurs is the oxidation of NADH to NAD+ and the reduction of pyruvate to lactate. To measure the rate of the chemical reaction, one can measure the disappearance of the substrate or the accumulation of one of the products. In the case of LDH, spectrophotometric methods (based on the light-absorbing properties of a substrate or product) are available to measure the rate of production of NAD+. The choice of substrate determines what enz)rme activity is to be measured. 

What enzyme catalyzes the reduction of pyruvate to lactate ... 

As in alcoholic fermentation, there is no net oxidation-reduction. The NADH formed in the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of pyruvate. The regeneration of NAD in the reduction of pyruvate to lactate or ethanol sustains the continued operation of glycolysis under anaerobic conditions. 

Fig. 19.11. Anaerobic glycolysis. Phosphate is transferred from high-energy intermediates of the pathway to ADP. Because NADH from the pathway is reoxidized by reduction of pyruvate to lactate, no oxygen is required.

When the oxidative capacity of a cell is limited (e.g., the red blood cell, which has no mitochondria), the pyruvate and NADH produced from glycolysis cannot be oxidized aerobically. The NADH is therefore oxidized to NAD in the cytosol by reduction of pyruvate to lactate. This reaction is catalyzed by lactate dehydrogenase (LDH) (Fig. 22.9). The net reaction for anaerobic glycolysis is ... 

Carbon-carbon multiple bonds can be hydrogenated using illumination of semiconductor particles as catalysts [152,153]. One example is the reduction of pyruvate to lactate under illumination on aqueous suspension of TiOj particles [154]. Triethanolamine increases the reaction efficiency by donating electrons to h. Alkyne reduction, however, usually results in a mixmre of alkenes and alkanes [152]. 

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