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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.

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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-1?. PGC-1? 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. 

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