Discuss the structure and coenzyme role of folic acid?
Folic acid participates in the transfer of single carbon units. Folate-dependent single-carbon transfer reactions are important in amino acid metabolism and in pathways leading to biosynthesis of DNA, RNA, membrane lipids, and neurotransmitters.
Structure of folic acid- Folic acid is a composite molecule, being made up of three parts: a pteridine ring system (6-methylpterin), para-amino benzoic acid, and Glutamic acid (Figure-1).
Figure-1- Structure and Reduction of folic acid
The glutamic acid doesn’t participate in the coenzyme functions of folic acid. Instead, folic acid in the interior of the cell may contain a “chain” of three to eight glutamic acid residues, which serves as a negatively charged “handle” to keep the coenzyme inside cells and/or bound to the appropriate enzymes. The pteridine portion of the coenzyme and the p-amino benzoic acid portion participate directly in the metabolic reactions of folate.
Reduction of folic acid-To carry out the transfer of 1-carbon units, NADPH must reduce folic acid twice in the cell. The pyrazine ring of the 6-methylpterin is reduced at each of the two N-C double bonds by folate reductase enzyme.
The resulting 5, 6, 7, 8-tetrahydrofolate is the acceptor of 1-carbon groups (Figure-1)
Forms of folic acid as one carbon carrier-Tetrahydrofolate can carry one-carbon fragments attached to N-5 (formyl, formimino, or methyl groups), N-10 (formyl) or bridging N-5–N-10 (methylene or methenyl groups).
Sources of one carbon fragments– The major point of entry for one-carbon fragments into substituted folates is methylene-tetrahydrofolate, which is formed by the reaction of glycine, serine, and choline with tetrahydrofolate. Serine is the most important source of substituted folates for biosynthetic reactions, and the activity of serine hydroxy methyltransferase is regulated by the state of folate substitution and the availability of folate. The reaction is reversible, and in liver it can form serine from glycine as a substrate for gluconeogenesis. One carbon fragments are also produced from the metabolism of Tryptophan and Histidine.
Utilization of one carbon fragments- Methylene-, methenyl-, and 10-formyl-tetrahydrofolates are inter convertible (Figure-2)
The N5,N10-methylene-tetrahydrofolate can either donate its single-carbon group directly, be oxidized by NADP to the methenyl form, or be reduced by NADH to the methyl form (See figure). Depending on the biosynthetic pathway involved, any of these species can donate the 1-carbon group to an acceptor. The methylene form donates its methyl group during the biosynthesis of thymidine nucleotides for DNA synthesis, the methenyl form donates its group as a Formyl group during purine biosynthesis, and the methyl form is the donor of the methyl group to sulfur during methionine formation. When one-carbon folates are not required, the oxidation of formyl-tetrahydrofolate to yield carbon dioxide provides a means of maintaining a pool of free folate.
Figure-2- showing the conversions of one-carbon units on Tetrahydrofolate.
Transfer of Methyl Group
Although Tetrahydrofolate can carry a methyl group at N-5, the methyl group’s transfer potential is insufficient for most biosynthetic reactions.
S-Adenosylmethionine (adoMet) is more commonly used for methyl group transfers. It is synthesized from ATP and methionine by the action of methionine Adenosyl transferase (See figure-3).
Methyl transfer from S-AdoMet is highly favored chemically and metabolically. First, transfer of the methyl group relieves a positive charge on the Sulfur of S-AdoMet. Secondly, the bond between the Sulfur and the 5′ carbon of the adenosine is rapidly hydrolyzed, leaving homocysteine and free adenosine.
Figure- 3-showing the synthesis of S- Adenosyl Methionine and remethylation of Homocysteine to form Methionine.
Homocysteine itself is converted to methionine by the transfer of a methyl group from N5-methyl-tetrahydrofolate to homocysteine, regenerating methionine. The methyl group of N5-methyl-tetrahydrofolate is derived from serine, originally, so the net effect of this pathway is to move methyl groups from serine to a variety of acceptors, including homocysteine, nucleic acid bases, membrane lipids, and protein side chains.
Q.2-What is “folate trap” or “methyl group trap”?
Why does vitamin B12 deficiency precipitate folic deficiency also?
What is the reason that folic and vitamin B12 deficiencies always coexist?
If megaloblastic anemia is treated by giving only folic acid the neurological symptoms of B12 deficiency worsen, why is it so?
Answer- The conversion of 5,10-methylene-THF into 5-methyl-THF, which is catalyzed by MTHFR (5,10-methylenetetrahydrofolate reductase), is irreversible. The only way to make further use of 5-methyl-THF and to maintain the folate cycle consists in the vitamin-B12-dependent remethylation of homocysteine to methionine (regenerating THF). The methyl group transfer is therefore greatly dependent on 5-methyl-THF and the availability of vitamin-B12. In humans, this is the only known direct link of the metabolism of two vitamins; folic acid and vitamin-B12 both need each other ( See figure 4 below).
Figure-4- showing the interdependence of folic acid and vitamin B12 and the methylation cycle.
In cases of vitamin-B12 deficiency, it is possible that, in spite of sufficient availability of folates (and 5-methyl-THF), an intracellular deficiency of biologically active THF arises. This situation is called a ‘folate trap’ (or methyl group trap) because, on one hand, the concentration of 5-methyl-THF continues to rise and on the other hand, due to it being prevented from releasing methyl groups, a ‘metabolic dead-end situation’ develops, which leads to the inevitable blockage of the methylation cycle. The co-factors for the C1-transfers decrease and replication as well as the cell division rate are reduced. Hence, the principal problem is the decreasing activity of methionine synthase under vitamin-B12 deficiency with secondary disorders affecting the folate metabolism and insufficient de-novo synthesis of purines and pyrimidines. There is therefore functional deficiency of folate, secondary to the deficiency of vitamin B12.
The deficiency in active folic acids first affects the quickly dividing and highly proliferating hematopoiesis cells in the bone marrow and can even lead to pancytopenia.
Clinically, there is no difference between vitamin-B12 deficiency anaemia and folic acid deficiency anaemia. If such anaemia is treated with vitamin-B12, the blockage is immediately stopped and the blood count quickly normalizes. However, if the anaemia is exclusively treated with folic acid, it is simply converted to dihydrofolate and THF.
Long-term therapy using high doses of folic acid could therefore conceal the real cause i.e. pernicious (vitamin B12-deficiency) anaemia for a long time. The serum folate continues to rise (congestion of non-regenerated 5-methyl-THF) while the intracellular folate concentration (erythrocytes) drops. This situation interrupts the methylation cycle with numerous cell processes, among them the synthesis of myelin, the nerve fiber lining, being blocked due to a deficiency of methyl groups. A long undetected (causal) vitamin-B12-deficiency can therefore result in serious neurological damage.
Exclusive folic acid therapy can thus lead to neurological damage or even cause serious damage progression.
Q.3-What are folate antagonists? What is their importance in the clinical field?
Answer- Folate antagonists were originally developed as antileukemic agents, but are now being used and/or investigated in the treatment of a wide range of cancerous and non-cancerous diseases. The clinical importance of some of the folate antagonists is as follows-
A) Folate antagonists used in non cancerous diseases
1) Sulfanilamide is the simplest of the sulfa drugs, used as antibacterial agents. The similarity of sulfanilamide to p-amino benzoic acid is shown in Figure-5. Because its shape is similar to that of p-aminobenzoic acid, sulfanilamide inhibits the growth of bacteria by interfering with their ability to use p-aminobenzoic acid to synthesize folic acid. Sulfa drugs were the first antimetabolites to be used in the treatment of infectious disease. Because humans don’t make folic acid, sulfanilamide is not toxic to humans in the doses that inhibit bacteria. This ability to inhibit bacteria while sparing humans made them useful in preventing or treating various infections.
Figure-5-Showing the structural similarity of Sulfanilamide and p-Amino benzoic acid
2) Trimethoprim and Pyrimethamine
The one-carbon fragment of methylene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme; inhibitors of these enzymes can be used as antibacterial drugs (eg, trimethoprim) and Antimalarial drugs (eg, pyrimethamine).
B) Folate antagonists used as anticancer drugs
a) Inhibitors of dihydrofolate reductase
1) Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anti-cancer
2) Aminopterin is also an inhibitor of DHFR enzyme and is used as an anticancer drug.
b) Inhibitors of Thymidylate synthase
The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA(Figure-6). Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division.
Figure-6- showing the action of thymidylate synthase. Thymidylate is synthesized by the Methylation of uridylate (dUMP) in a reaction catalyzed by the enzyme Thymidylate synthase. This reaction requires a methyl donor and a source of reducing equivalents, which are both provided by N5, N10-methylene THF). For this reaction to continue, the regeneration of THF from Dihydro folate (DHF) must occur.
1) The antagonist 5-fluorouracil acts to indirectly inhibit the enzyme thymidylate synthase. The primary effect of TS inhibition by 5-FU is a depletion of dTMP and dTTP levels, resulting in inhibition of DNA synthesis and “thymine-less death.” Thymine-less death occurs when a cell can still synthesize RNA and protein, but is unable to make DNA resulting in cell overgrowth, and later, death.
2) Thymitaq (TM), or Nolatrexed dichloride (also referred to as AG337), is a noncompetitive inhibitor, of Thymidylate Synthase.
All these inhibitors are used as anticancer drugs.
Trimetrexate, Lometrexol and Pemetrexed are also some of the upcoming folate antagonists used in cancer chemotherapy.
Q.4-Why do the cancer patients on Methotrexate therapy develop glossitis, oral ulcers and diarrhea? How can these symptoms be treated?
Answer- Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anti-cancer drug. Methotrexate blocks the cell’s ability to regenerate THF, leading to inhibition of these biosynthetic pathways. The lack of nucleotides prevents DNA synthesis, and these cancer cells cannot divide without DNA synthesis.
Unfortunately, the effects of Methotrexate are nonspecific and other rapidly dividing cells such as epithelial cells in the oral cavity, intestine, skin, and blood cells are also inhibited. This leads to the side effects associated with methotrexate (and other cancer chemotherapy drugs) such as mouth sores, low white blood cell counts, stomach upset, hair loss, skin rashes, and itching. Less frequent adverse effects include reversible increases in transaminases and hypersensitivity-like pulmonary syndrome. Chronic low-dose methotrexate can cause hepatic fibrosis.
Leucovorin (LV) is a form of folic acid that can help “rescue” or reverse the toxic effects of methotrexate. LV is not a folate antagonist per se, but the folate derivative 5-formyltetrahydrofolate, After transport through the membrane,LV is metabolized to 5,10-MTHF, thus increasing the availability of 5,10-MTHF. Through this mechanism, it modulates the actions of many folate antagonists.LV can also enhance the antitumor activity of some folate antagonists.
N5-formyl THF is normally administered 24 hours following treatment with methotrexate; it can be converted to THF by these normal cells by bypassing the block caused by methotrexate. Therefore, these normal cells can synthesize deoxy thymidine and carry out DNA synthesis.
Please help "Biochemistry for Medics" by CLICKING ON THE ADVERTISEMENTS above!