Hypoxanthine-guanine phosphoribosyltransferase
Encyclopedia
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is an enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
encoded in humans by the HPRT1 gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
.
HGPRT is a transferase
Transferase
In biochemistry, a transferase is an enzyme that catalyzes the transfer of a functional group from one molecule to another . For example, an enzyme that catalyzed this reaction would be a transferase:In this example, A would be the donor, and B would be the acceptor...
that catalyzes conversion of hypoxanthine
Hypoxanthine
Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-Hydroxypurine. Hypoxanthine is a necessary additive in certain cell,...
to inosine monophosphate and guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
to guanosine monophosphate
Guanosine monophosphate
Guanosine monophosphate, also known as 5'-guanidylic acid or guanylic acid and abbreviated GMP, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid with the nucleoside guanosine. GMP consists of the phosphate group, the pentose sugar ribose, and the nucleobase...
. This reaction transfers the 5-phosphoribosyl group from 5-phosphoribosyl 1-pyrophosphate to the purine. HGPRT plays a central role in the generation of purine
Purine
A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s through the purine salvage pathway.
Function
HGPRT catalyzes the following reactions:| Substrate | Product | Notes >- | hypoxanthine Hypoxanthine Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-Hydroxypurine. Hypoxanthine is a necessary additive in certain cell,... |
inosine monophosphate | >- | guanosine monophosphate Guanosine monophosphate Guanosine monophosphate, also known as 5'-guanidylic acid or guanylic acid and abbreviated GMP, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid with the nucleoside guanosine. GMP consists of the phosphate group, the pentose sugar ribose, and the nucleobase... |
>- | xanthosine monophosphate Xanthosine monophosphate Xanthosine monophosphate is an intermediate in purine metabolism. It is a ribonucleoside monophosphate. It is formed from IMP via the action of IMP dehydrogenase, and it forms GMP via the action of GMP synthase.-Clinical significance:... |
Only certain HPRTs. |
HGPRTase functions primarily to salvage purines from degraded DNA to reintroduce into purine synthetic pathways. In this role, catalyzes in the reaction between guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
and phosphoribosyl pyrophosphate
Phosphoribosyl pyrophosphate
Phosphoribosyl pyrophosphate is a pentosephosphate.It is formed from ribose 5-phosphate by the enzyme ribose-phosphate diphosphokinase.It plays a role in transferring phospho-ribose groups in several reactions:...
(PRPP) to form GMP
Guanosine monophosphate
Guanosine monophosphate, also known as 5'-guanidylic acid or guanylic acid and abbreviated GMP, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid with the nucleoside guanosine. GMP consists of the phosphate group, the pentose sugar ribose, and the nucleobase...
.
Role in disease
Mutations in the gene lead to hyperuricemiaHyperuricemia
Hyperuricemia is a level of uric acid in the blood that is abnormally high. In humans, the upper end of the normal range is 360 µmol/L for women and 400 µmol/L for men.-Causes:...
:
- Some men have partial (up to 20% less activity of the enzyme) HGPRT deficiency that causes high levels of uric acidUric acidUric acid is a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen with the formula C5H4N4O3. It forms ions and salts known as urates and acid urates such as ammonium acid urate. Uric acid is created when the body breaks down purine nucleotides. High blood concentrations of uric acid...
in the blood, which leads to the development of gouty arthritis and the formation of uric acid stones in the urinary tract. This condition has been named the Kelley-Seegmiller syndrome. - Lesch-Nyhan syndromeLesch-Nyhan syndromeLesch–Nyhan syndrome , also known as Nyhan's syndrome, Kelley-Seegmiller syndrome and Juvenile gout, is a rare inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase , produced by mutations in the HPRT gene located on X chromosome. LNS affects about...
is due to HPRT mutations resulting in extremely ineffective enzyme activity. - Some mutations have been linked to goutGoutGout is a medical condition usually characterized by recurrent attacks of acute inflammatory arthritis—a red, tender, hot, swollen joint. The metatarsal-phalangeal joint at the base of the big toe is the most commonly affected . However, it may also present as tophi, kidney stones, or urate...
, the risk of which is increased in hyperuricemia.
Application to hybridomas
B cells contain this enzyme, which enables them to survive when fused to myeloma cells when grown on HAT mediumHAT medium
HAT Medium is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase, with hypoxanthine and thymidine which are intermediates in DNA synthesis...
to produce monoclonal antibodies
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
. The antibodies are produced from cells called hybridoma
Hybridoma
Hybridoma technology is a technology of forming hybrid cell lines by fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis...
cells. A hybridoma, which can be considered as a hybrid cell, is produced by the injection of a specific antigen into a mouse, procuring the antibody-producing cell from the mouse's spleen and the subsequent fusion of this cell with a cancerous immune cell called a myeloma cell. The hybrid cell, which is thus produced, can be cloned to produce many identical daughter clones. These daughter clones then secrete the immune cell product.
The method of selecting hybridomas is by use of HAT medium
HAT medium
HAT Medium is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase, with hypoxanthine and thymidine which are intermediates in DNA synthesis...
, which contain hypoxanthine
Hypoxanthine
Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-Hydroxypurine. Hypoxanthine is a necessary additive in certain cell,...
, aminopterin
Aminopterin
Aminopterin , a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase...
, and thymidine
Thymidine
Thymidine is a chemical compound, more precisely a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine in double-stranded DNA...
. The aminopterin inhibits enzyme dihydrofolate reductase
Dihydrofolate reductase
- Function :Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids...
(DHFR), which is necessary in the de novo synthesis
De novo synthesis
De novo synthesis refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to their being recycled after partial degradation. For example, nucleotides are not needed in the diet as they can be constructed from small precursor molecules such as...
of nucleic acids. Thus, the cell is left with no other option but to use the alternate salvage pathway
Salvage pathway
A salvage pathway is a pathway in which nucleotides are synthesized from intermediates in the degradative pathway for nucleotides....
, which utilises HGPRT. In the HAT medium, HGPRT- cell lines will die, as they cannot synthesise nucleic acids through salvage pathway. Only HGPRT+ cells will survive in presence of aminopterin, which are the hybridoma cells and plasma cells. The plasma cells eventually die as they are mortal cell lines, thus only hybridoma cells are left surviving. The hybrid cell (hybridoma cell) can be cloned to produce many identical daughter clones. These daughter clones subsequently secrete the monoclonal antibody product.