LDH

Lactate Dehydrogenase (LDH, EC 1.1.1.27), also known as NAD⁺ oxidase, catalyzes the reversible conversion of pyruvate and L-lactic acid and is an important oxidoreductase in the process of glycolysis, which is widely found in animal tissues and various microbial cells. The lactic acid is a chiral molecule with two configurations of D-lactic acid and L-lactic acid. The human body can only metabolize and utilize L-lactic acid. Therefore, the World Health Organization has advocated the use of L-lactic acid as a food additive and internal medicine to replace the currently widely used DL-lactic acid. In addition L-lactic acid, L-lactate and its polymers are also widely used in medicine, agriculture and chemical industry. People also use poly-L-lactic acid to produce biodegradable plastics, green packaging materials and household films to solve the increasingly serious environmental pollution problems.

Sources

Lactate dehydrogenase is widely present in animal tissues such as myocardium, liver, kidney, skeletal muscle, pancreas, and lung. Animal LDH is a tetrameric molecule with a molecular weight of 130000 to 145000. It belongs to the isozyme family and has five molecular forms. Two of the forms are composed of the same subunit. These two LDHs are H₄ mainly distributed in in myocardium and M₄ mainly distributed in skeletal muscle. The other three LDH consist of mixed subunits, they are H₃M, H₂M₂, and HM₃. In any form of LDH, each H or M subunit contains an active catalytic center. For tetramers of mixed subunits, such as H₃M, the catalytic kinetic properties are the same as the 3:1 mixture of H₄ and M₄, indicating that the active centers of the same subunits in the different types of LDH molecules are very similar, and there is no significant interaction between the catalytic reactions on each subunit.

Bacterial lactate dehydrogenase can be divided into two types: one is LDH with NAD⁺ as coenzyme (NAD-dependent LDH, nLDH), and the other is LDH with cytochrome c, FAD or FMN, which belongs to non-dependent NAD⁺ type (NAD-independent LDH, iLDH). iLDH is present in both anaerobic and aerobic bacteria that catalyze lactic acid to produce pyruvate, which does not require NAD⁺/NADH as a coenzyme. No in vivo and in vitro experiments have detected that the enzyme can catalyze the occurrence of a reverse reaction. nLDH is located in the cytoplasm and is the key enzyme in the production process of Embden–Meyerhof–Parnas (EMP pathway). It is mainly used to catalyze pyruvic acid to produce lactic acid. The activity of nLDH is affected by the oxygen supply of bacteria. Certain bacteria nLDH bind to lactic acid and NAD under alkaline conditions. LDH in microbial cells did not find isoenzymes, and its conservation was far inferior to that of animal tissues. The LDH properties of different strains were quite different.

Catalytic Mechanism

L-lactate dehydrogenase reversibly catalyzes the oxidation of L-lactate to produce pyruvate, using the NAD⁺/NADH as a coenzyme. In this reaction, each subunit of LDH binds a substrate molecule and a coenzyme molecule, respectively, and react independently. In the positive reaction, LDH, with the aid of NAD⁺, reversibly catalyzes the oxidation of deprotonated lactic acid to produce deprotonated pyruvic acid and H⁺. At the same time, NAD⁺ binds a H⁺ from the substrate to form NADH. The reaction can be reversed. The reaction equation is: pyruvate + reduced coenzyme I + H⁺ → lactic acid + NAD⁺. E. Silverstein et al. demonstrated that the catalytic mechanism of LDH catalytic reactions are as follows: Firstly, the protease binds to the coenzyme (NADH) as a binary complex. At the same time, the conformation of the enzyme changes, which binds to the substrate pyruvic acid. During the reaction, the product lactic acid is released, then, the enzyme is separated from the oxidized coenzyme (NAD⁺). The H⁺ generated in the reaction is transferred to the enzyme residue (His-195) and released after completion of the reaction.

Applications

LDH has important value when used to analyze other enzymes and biological products. LDH is a general-purpose enzyme that detects other enzymes such as alanine aminotransferase (ALT), ATPase, myokinase, and pyruvate kinase. ALT is a commonly used clinical diagnostic enzyme. When the lesion occurs in the tissue, a large amount of ALT in the cell is released into the blood, which increases the activity of ALT in the serum. The principle of reaction for determining ALT activity by LDH is: L-alanine + α-ketoglutarate glutaric → pyruvate + L-glutamic acid; pyruvic acid + NADH + H⁺ → L-lactic acid + NAD⁺ + H₂O. The reduction of NADH causes a decrease in absorbance at a wavelength of 340nm, which is proportional to the ALT activity, thereby detecting whether the tissue is diseased. The purity of LDH directly affects the accuracy of the determination of ALT activity. In addition, LDH also plays a very important role in the determination of triglycerides.

At present, LDH can also be used in nanotechnology. By immobilizing LDH on the surface of nanogold-cysteamine-modified gold electrode, a novel nano-biomimetic functional interface can be constructed. The novel enzyme biosensor based on this functional interface exhibits a good linear relationship to the electrocatalytic response of lactic acid, and has high sensitivity and selectivity for the determination of lactic acid, which has a good application prospect in clinical and biological fields. In addition, the application of LDH kit to detect NK cell activity has also been applied. This method eliminates the cumbersome procedures such as provisional reagents and can be used for automatic analyzers to measure batch standards.