Lactate Dehydrogenase In Anaerobic Cellular Respiration
Abstract
Lactate dehydrogenase is a vital enzyme in the process of anaerobic cellular respiration. Anaerobic cellular respiration is an important function in plants, animals, and bacteria to produce ATP. Lactate dehydrogenase is found in almost all living cells to serve as a catalyst for anaerobic cellular respiration.
Introduction
The objective of studying lactate dehydrogenase is to learn about the structure, function, and importance. The goal is to become familiar with the enzyme and the metabolic processes that it is involved with. Without lactate dehydrogenase anaerobic cellular respiration would not occur. This would substantially inhibit the production of ATP by the cell. Some bacteria rely solely on anaerobic cellular respiration as the main source of ATP, and without lactate dehydrogenase there would be no energy production in the cell.
Metabolic Pathway of Lactate Dehydrogenase
Cellular respiration that does not require oxygen is defined as anaerobic cellular respiration. A final acceptor is needed at the end of the electron transport chain. The final acceptor in aerobic cellular respiration is oxygen, but in anaerobic cellular respiration the final acceptor is a less-oxidizing compound. Less energy is formed from each oxidized molecule since these molecules have a smaller reduction potential than oxygen. Anaerobic cellular respiration is much less efficient when compared to aerobic cellular respiration. Anaerobic cellular respiration functions to produce lactate acid from pyruvate with no oxygen present. Anaerobic cellular respiration is important for glycolysis. The accumulation of pyruvate would slow down ATP production. Anaerobic cellular respiration functions to regenerate NAD+ from NADH. In humans, as one exercises, glucose is completely broken down which releases carbon atoms as carbon dioxide and hydrogen molecules as water. This process requires substantial amounts of oxygen. Energy production will stop at the end of glycolysis if the supply of oxygen does not meet the demand for oxygen. Energy can still be produced when the supply of oxygen does not meet the demand through anaerobic cellular respiration. However, this process is less efficient and less ATP is produced. Lactate dehydrogenase makes this process possible.
Lactate dehydrogenase is a key enzyme that is involved with anaerobic cellular respiration. As stated above anaerobic cellular respiration is key in the regeneration of NAD+ from NADH. Lactate dehydrogenase is the main enzyme involved with converting NADH to NAD+. As this conversion is occurring, lactate dehydrogenase also converts lactate to pyruvic acid and back. During glycolysis, the hydrogen atom from glucose is put on NAD+ and forms NADH. These hydrogen atoms are transferred to oxygen to form water when oxygen is available, however, when oxygen is unavailable, the NADH will build up and there is not enough NAD+ to continue producing ATP using glycolysis. Lactate dehydrogenase combines pyruvate and the built up NADH to form lactic acid and NAD+. This NAD+ formed can then be used to complete another cycle of glycolysis, thus producing more ATP. This process quickly creates more energy.
The Gene Ontology Terms
The biological processes for lactate dehydrogenase according to gene ontology are vast. The biological processes of lactate dehydrogenase include: response to hypoxia, carbohydrate metabolic process, lactate metabolic process, pyruvate metabolic process, glycolytic metabolic process, response to nutrient, response to glucose, response to organic cyclic compound, NAD metabolic process, carboxylic acid metabolic process, response to drug, response to hydrogen peroxide, positive regulation of apoptotic process, response to estrogen, post-embryonic animal organ development, response to cAMP, and oxidation-reduction process. Lactate dehydrogenase can be found throughout the cell. According to gene ontology lactate dehydrogenase is found in the following locations in the cell: nucleus, cytoplasm, cytosol, membrane, and integral component of membrane. It has been seen that LDH has many molecular functions. Some of the molecular functions are: catalytic activity, lactate dehydrogenase activity, L-lactate dehydrogenase activity, protein binding, oxidoreductase activity, acting on the CH-OH groups of donors, NAD or NADP as an acceptor, kinase binding, identical protein binding, cadherin binding, and NAD binding.
History of Isolation
Human LDH-X was isolated from frozen samples of semen using affinity chromatography. When NAD+ is present the LDH-X does not bind to AMP-Sepharose. The other lactate dehydrogenase isoenzymes will bind to AMP-Sepharose. This is the key point in isolating LDH-X versus the other isoenzymes.
The frozen semen samples were thawed and centrifuged at 30,000 g and four degrees Celsius for 20 minutes. Approximately 500 milliliters of the seminal fluid were separated by ammonium sulfate. The precipitate that was formed was dialyzed against a sodium phosphate buffer. The sodium phosphate buffer had a pH of 6. 8. This same buffer was used for all of the chromatography steps. The temperature was kept at 4 degrees Celsius for the entire procedure. In the presence of NADH, lactate dehydrogenase isoenzymes will bind to the column and are then eluted by the buffer. In the presence of buffer only, lactate dehydrogenase isoenzymes will also bind to AMP-Sepharose. It was found that if equal volumes of seminal fluid and buffer containing NADH were mixed immediately before loading it into the column, enough NADH was still present to allow complete binding of lactate dehydrogenase to the column. AMP-Sepharose was used to separate LDH-X from the other LDH isoenzymes since LDH-X does not bind to AMP-Sepharose.
Characteristics of the Protein
The lactate dehydrogenase contains a disordered portion of approximately 50 residues. This disordered region has discontinuous electron density. The current model of the lactate dehydrogenase protein contains: residues 9-328, 375-567, an acetate molecule, a FAD molecule, and approximately 200 water molecules for each monomer. The two monomers are basically identical.
The structure of the lactate dehydrogenase protein is made up of three discontinuous domains: the FAD-binding domain (residues 1–268 and 520–571), the cap domain (residues 269–310, 388–425, and 450–519), and the membrane-binding domain (residues 311–387 and 426–449, residues 329 –376 are in the disordered region). The FAD-binding domain contains two alpha + beta subdomains. One is made up of three antiparallel beta beta strands surrounded by five alpha helices and is packed against the second domain. The second subdomain is made up of five parallel beta strands surrounded by four alpha helices. The cap domain is composed of a large seven stranded antiparallel beta sheet flanked on both sides by alpha helices. The membrane binding domain is made up of four alpha helices. The largest difference between these structures is in the membrane-bounding domain.
Lactate dehydrogenase is considered to be a part of the FAD-containing family. The main difference between LDH and other members of the FAD-containing family is the membrane binding domain. In other proteins of the FAD-containing family, the membrane binding domain is either absent or significantly different. The lactate dehydrogenase membrane binding domain has an electropositive surface with six Arg and five Lys residues. This is expected to interact with the negatively charged phospholipid head groups of the membrane. Based on this observation, lactate dehydrogenase binds to the membrane with electrostatic forces rather than hydrophobic forces. Some other members of the FAD-containing protein family are: vanillyl-alcohol oxidase, p-cresol methylhydroxylase (PCMH), and UDP-N-acetylenolpyruvyglucosamine (MurB). The proteins in this family can be found in both eukaryotes and eubacteria.
Characteristics of the Gene for Lactate Dehydrogenase
The LDHA gene in humans is located on chromosome 11p15. 4. Chromosome 11 is approximately 135 million base pairs and accounts for around 4-4. 5 percent of DNA in the cells. Chromosome 11 contains approximately 1,300-1,400 genes that give instructions for synthesizing proteins. These proteins have a wide array of tasks in the body. The LDHB gene is located on chromosome12p12. 2-p12. 1. Chromosome 12 is made up of almost 134 million base pairs and accounts for around 4-4. 5 percent of the DNA in cells. Chromosome 12 contains approximately 1,100-1,200 genes that provide instructions for synthesizing proteins. These proteins also also have a wide array of tasks in the body. The LDHC gene is only expressed in the testes and can be found on chromosome 11p15. 5-p15. 3. The human genome also has several non-transcribed LDHA pseudogenes. M subunit mutations have been observed to be disease causing, H subunit mutations have not been linked to a certain disease causing trait. LDHA mutations have been linked to cause exertional myoglobinuria and Fanconi-Bickel Syndrome.
There are four genes for lactate dehydrogenase: LDHA, LDHB, LDHC, and LDHD. LDHA, LDHB, and LDHC are the L-isomers. LDHD is a D-isomer. The L-isomers use and produce L-lactate. L-lactate is the major enantiomer found in vertebrates. LDHA is called the M subunit and is mostly found in skeletal muscle. LDHB is called the H subunit and is mostly found in the heart. Five isoenzymes can be formed from the M and H subunits of LDH. The isoenzymes are: LDH-1 (4H), LDH-2 (3H, 1M), LDH-3 (2H, 2M), LDH-4 (1H, 3M), and LDH-5 (5M). LDH-1 and LDH-5 have the same active site region. These isoenzymes are similar in function but have a different distribution throughout tissues.
Regulation of the Enzyme at Transcriptional and Enzymatic Levels
The LDHA promoter region is well known to contain the consensus sequences for, and be regulated by, major transcription factors: hypoxia-inducible factor 1 (HIF1) and c-Myc. Forkhead box protein M1 (FOXM1) and Kruppel-like factor 4 (KLF4) are identified as transcriptional regulators of LDHA. The regulation of LDHA is very complex. Complete understanding of how LDHA is regulated is far from being achieved. It has also been found that LDHA transcription is influenced by other factors such as: lactate, cyclic adenosine monophosphate (cAMP), estrogen, ErB2, and heat shock factor. It is highly likely that transcriptional regulation of LDHA is influenced by many other unknown factors. Like many other known enzymes, the post-transcriptional activity of LDHA is regulated by the phosphorylation and acetylation of amino acid residues. LDH also undergoes transcriptional regulation by PGC-1α. PGC-1α regulates LDH by decreasing LDHA mRNA transcription and the enzymatic activity of pyruvate to lactate conversion.
At the enzymatic level, LDH is regulated by the relative concentrations of its substrates. During times of major muscular output LDH becomes more active because of an increase in substrates for the LDH reaction. When the muscles are forced to produce a large amount of power, the demand for ATP causes a buildup of free ADP, AMP, and Pi. The resulting glycolytic flux, makes it difficult for shuttle enzymes to metabolize pyruvate. The flux through LDH increases in response to increased levels of pyruvate and NADH to metabolize pyruvate into lactate.
Conclusion
There are many more processes lactate dehydrogenase is believed to be involved with. This enzyme will continue to be further studied in hopes of being targeted for certain disorders. Recent research has shown lactate dehydrogenase to be a therapeutic target for certain types of cancers. This gives hope that lactate dehydrogenase could be a potential target for the treatment of cancers and cancer associated disorders. There are vast pharmacological applications to be considered from this research. It can be seen how important lactate dehydrogenase is in the cell.
