Lactate dehydrogenase reaction catalyzed

The biochemist needs a test, called an assay, for some unique identifying property of the protein. A positive result on the assay indicates that the protein is present. Determining an effective assay is often difficult but, the more specific the assay, the more effective the purification. For enzymes, which are protein catalysts (Chapter 8), the assay usually measures enzyme activity—that is, the ability of the enzyme to promote a particular chemical reaction. This activity is often measured indirectly. Consider the enzyme lactate dehydrogenase, which catalyzes the following reaction in the synthesis of glucose ... 

Fig. 18. Lactate dehydrogenases can catalyze the oxidation of glyoxalate to oxalate, as shown in the top reaction, and also the reduction of glyoxalate to glycolic acid (not shown). Note that glyoxalate differs from lactate by the isosteric replacement of the methyl group by a hydroxyl.

Lactate dehydrogenase (LDH) catalyzes the reaction below. This reaction provides an important source of NAD+ for cells undergoing anaerobic glycolysis. 

Fig. 22.9. Lactate dehydrogenase reaction. Pyruvate, which may be produced by glycolysis, is reduced to lactate. The reaction, which occurs in the cytosol, requires NADH and is catalyzed by lactate dehydrogenase. This reaction is readily reversible.

Determination of LDH Lactate dehydrogenase (LDH) catalyzes the equilibrium reaction of pyruvate to lactate. The activity of serum LDH is due to the presence of the enzyme released from damaged organs and tissues such as liver, heart, skeletal muscle, erythrocytes, etc. because LDH is located in the cytoplasm of the cells. Therefore, the activity of LDH is useful for screening for the existence of cell injuries, estimation of damaged tissues, and evaluation of treatment of diseases. LDH has five isoenzymes, and their patterns are of diagnostic importance. 

The absorbance change (AM) at 340 nm can be used to determine the amount of pymvate remaining. The lactate dehydrogenase [9001-60-9] catalyzed reaction can also be used in the reverse direction to measure lactate. The reaction takes place in a buffer of pH 9—10 that neutralizes Hberated H". ... 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

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. 

Kinetic Constants for a Reaction Catalyzed by Lactate Dehydrogenase... 

We know that anaerobic glycolysis of glncose yields pyruvate and/or lactate, interconvertable metabolites. Pyruvate is converted into acetyl-SCoAin the following reaction, catalyzed by the pyruvate dehydrogenase complex ... 

A sequential enzyme-catalyzed reaction mechanism in which two substrates react to form two products and in which there is a preferred order in the binding of substrates and release of products. Several enzymes have been reported to have this type of binding mechanism, including alcohol dehydrogenase , carbamate kinase , lactate dehydrogenase , and ribitol dehydrogenase. ... 

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. 

They stated further that, the new adaptive enzyme catalyzing Reaction 3 appears to be similar to the malic enzyme of pigeon liver, although strictly DPN (instead of TPN)-specific. The coenzyme specificity explains the ready occurrence of Reaction 1. Therefore, the authors showed that exogenous NAD was required for the overall reaction (malic acid -> lactic acid), but because this activity was measured manometrically, they never demonstrated the formation of reduced NAD. Similarly, they did not attempt to show that pyruvic acid was the intermediate between L-malic acid and lactic acid. Instead, the formation of pyruvic acid was inferred from the NAD requirement and because the malic acid dissimilation activity remained constant during purification while the lactate dehydrogenase activity decreased (14). In fact, attempts to show any appreciable amounts of pyruvic acid intermediate failed (22). 

The reactions of Eq. 15-19 occur nonenzymatically only under the influence of strong base but dehydrogenases often catalyze similar condensations relatively rapidly and reversibly. Pyruvate inhibits lactate dehydrogenase, 2-oxoglutarate inhibits glutamate dehydrogenase, and ketones inhibit a short-chain alcohol dehydrogenase in this manner.133,693... 

Enzymes in the cross-linked crystal form are essentially impervious to degradation by exogenous proteases and from autolysis, in the case of CLCs of proteases themselves [5], This stability makes the enzyme-catalyzed preparation of peptides and peptide mimics truly practical [6], Examples will be discussed in more detail in Sec. IV. Further, one could conceive of using multiple enzymes in one-pot reaction systems mimicking natural biosynthetic cascades. Indeed, the application of this concept has been reported for a mixture of lipoamide dehydrogenase and lactate dehydrogenase [19],... 

Isoenzymes Isoenzymes are different forms of an enzyme which catalyze the same reaction, but which exhibit different physical or kinetic properties. The isoenzymes of lactate dehydrogenase (LDH) can be separated electrophoretically and can be used clinically to diagnose a myocardial infarction. 

Quantitative Assay for Lactate Dehydrogenase The muscle enzyme lactate dehydrogenase catalyzes the reaction... 

Enzymatic Reaction Mechanisms I Lactate dehydrogenase is one of the many enzymes that require NADH as coenzyme. It catalyzes the conversion of pyruvate to lactate ... 

Answer The measurement of the activity of alanine aminotransferase by measurement of the reaction of its product with lactate dehydrogenase is an example of a coupled assay. The product of the transamination (pyruvate) is rapidly consumed in the subsequent indicator reaction, catalyzed by an excess of lactate dehydrogenase. The dehydrogenase uses the cofactor NADH, the disappearance of which is conveniently measured by observing the rate of decrease in NADH absorption at 340 nm. Thus, the rate of disappearance of NADH is a measure of the rate of the aminotransferase reaction, if NADH and lactate dehydrogenase are added in excess. 

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