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.
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
respiration is a form of cellular respiration that does not require the use of oxygen.
In the electron transport chain, a final acceptor at the end of the chain is necessary.
In aerobic respiration this final acceptor is oxygen, however, in anaerobic
respiration this 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, resulting in a much less efficient process
than aerobic cellular respiration. The function of anaerobic cellular
respiration is to convert pyruvate into lactate acid without oxygen. This
process is very important for glycolysis. ATP production would be very slow if
pyruvate were to accumulate. Anaerobic cellular respiration also allows for the
regeneration of 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 a substantial amount of oxygen. Energy
production will stop at the end of glycolysis if the supply of oxygen does not
meet the demand for oxygen. Through anaerobic cellular respiration, although
with much less efficiency, energy can still be produced. Without lactate
dehydrogenase this would be impossible.
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. When oxygen is available,
these hydrogen atoms are transferred to oxygen to form water. When oxygen is
unavailable, the NADH will build up and there is not enough NAD+ to continue
using glycolysis to produce ATP. 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
frozen human semen samples were thawed and centrifuged at 30000 g and four
degrees Celsius for 20 minutes. Approximately 500 milliliters of the seminal
fluid were separated by ammonium sulfate. The precipitate recovered was
dialyzed against a sodium phosphate buffer with 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
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 contains 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.
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 commonly called
the M subunit and is mostly found in skeletal muscle. LDHB is commonly 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
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-1?. PGC-1? regulates LDH by decreasing LDHA
mRNA transcription and the enzymatic activity of pyruvate to lactate conversion.
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
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.