methyleneH4folatemethylH4folate formylH4folate

Metabolic Pathways

Methylation regulates gene expression and maintains the differentiated state. The immediate methyl donor in most reactions is S- adenosyl methionine, or SAM. The waste product is S- adenosyl homocysteine. This is converted back to methionine through a series of reactions involving vitamin B12 and tetrahydrofolate. Once the homocysteine has been re-methylated, the methionine can be used again. Folates: folate → dihydrofolate → tetrahydrofolate (THF) ↔ methylene-THF → methyl-THF Folate Balance Vitamin B12 is the only acceptor of methyl-THF. There is also only one acceptor for methyl- B12 which is homocysteine in a reaction catalyzed by homocysteine methyltransferase. This is important because a defect in homocysteine methyltransferase or a deficiency of B12 can lead to a methyl-trap of THF and a subsequent deficiency. Thus, a deficiency in B12 can generate a large pool of methyl-THF that is unable to undergo reactions and will mimic folate deficiency. Another form of THF, formyl-THF or folinic acid) results from oxidation of methylene-THF or is formed from formate donating formyl group to THF.

Methotrexate An anti-folate Methotrexate competitively and irreversibly inhibits dihydrofolate reductase (DHFR), an enzyme in tetrahydrofolate synthesis. The prototypic antifol DHFR inhibitor is a 4-amino–substituted pterin compound. The substitution of an amino moiety for 4-hydroxyl results in a folate analogue with several thousand-fold increase in affinity for DHFR. The Ki of MTX for DHFR is below M, well below the Km of the natural substrate, dihydrofolate, which is in the micromolar range. It is this remarkable increase in affinity compared with the natural substrate that resulted in MTX being considered a “stoichiometric inhibitor” of DHFR. Folic acid is needed for the de novo synthesis of thymidine (DNA), for purine base synthesis, so all purine synthesis will be inhibited. Methotrexate, therefore, inhibits the synthesis of DNA, RNA, thymidylates, and proteins. Methotrexate acts specifically during DNA and RNA synthesis, and thus it is cytotoxic during the S-phase of the cell cycle. It therefore has a greater toxic effect on rapidly dividing cells which replicate their DNA more frequently, and thus inhibits the growth and proliferation of these non-cancerous cells as well as causing the side effects listed below. Structure similarity of folic acid (left) and methotrexate (right)

Oxidative addition Reductive elimination B12 - Cobalamin

Monsanto Acetic Acid Catalysis (organometallic catalysis in industry)

The Major Pterin Players Neopterin: biochemical precursor; immune response marker Folate/Folic acid: One carbon (C1) pool Biopterin: redox cofactor for (1) aromatic aminoacid hydroxlases (neurotransmitters) (2) NOS (nitric oxide synthase), regulator, signaling Methanoopterin: C1 chemistry (methyl transfer) methanogenesis Molybdopterin: O atom chemistry Synthetic pterins: methotrexate anti-folates

GTP Guanosine triphosphate Neopterin Biopterin  PAH cofactor NOS cofactor Methopterin Molybdopterin  Mo / W enzymes …molybdopterin biosynthesis proceeds via a pathway unlike that of any known pteridine biosynthetic pathway. Folate  C1 chemistry X How they’re related (or not)

Folates Folic acid (vitamin B9), tetrahydrofolate (cofactor), dihydrofolate, Etymology: Folate and folic acid derive from the Latin word folium, "leaf” C1 chemistry: methylation (a)Homocysteine to methionine; (b) uracil to thymine; (c) purine synthesis Nucleic acid metabolism involve the addition of C1 units. C1 units are derived from the breakdown of serine and glycine and carried by the coenzyme tetrahydrofolate (H 4 F), which is synthesized from folic acid in the diet. These C1 fragments can be oxidized and reduced while bound to the H 4 F coenzyme, so they may be inserted into the receiving molecules as formyl groups (most oxidized), hydroxymethyl or methyl groups (most reduced). [search Expasy[search Expasy: tetrahydrofolate] Methylene-THF (CH2FH4) is formed from THF by the addition of methylene groups from one of three carbon donors: formaldehyde, serine, or glycine. Methyl tetrahydrofolate (CH3-THF) can be made from methylene-THF by reduction of the methylene group with NADPH. Histidine can donate a single carbon to THF to form methenyl-THF.

Folates Folic acid (vitamin B9), tetrahydrofolate (cofactor), dihydrofolate, Etimology: Folate and folic acid derive from the Latin word folium, "leaf” C1 chemistry: methylation (a)Homocysteine to methionine; (b) uracil to thymine; (c) purine synthesis Health Lack of dietary folic acid leads to folate deficiency (FD). Resulting health problems: (1)neural tube defects in developing embryos. Neural tube closure takes place during early embryogenesis and requires interactions between genetic and environmental factors. Failure of neural tube closure is a common congenital malformation that results in morbidity and mortality. A major clinical achievement has been the use of folic acid supplements, which prevents ~50–75% of cases of neural tube defects. However, the mechanism underlying the beneficial effects of folic acid is far from clear. Biochemical, genetic and epidemiological observations have led to the development of the methylation hypothesis, which suggests that folic acid prevents neural tube defects by stimulating cellular methylation reactions. (2) homocysteine accumulation as a result of one carbon metabolism mechanism being impaired. (3) DNA synthesis and repair are impaired and this could lead to cancer development. A 2003 opinion article in the New York Times: Adding folic acid and micronutrients to the food supply of developing countries would have a greater impact than any other single action in improving world health.

Methanopterin A cousin to the folates, does C1 chemistry. A cofactor in methanogenesis, a bacterial process of methane production in respiration. Differs from folates in that phenyl group lacks a carbonyl, so is harder to reduce. [search Espasy[search Espasy: methanopterin]

Biopterin THB site Redox cofactor as tetrahdrobiopterin First discovered as cofactor (S. Kaufman) for PAH, phenylalanine hydroxylase; role = activation of O 2 for para-hydroxylation of phenyl group Later revealed as cofactor for analogous hydroxylations of tyrosine (to L-DOPA), tryptophan (to 5-hydroxy-tryptophan ). The class of enzymes is called the aromatic amino acid hydroxylases. These are precursors to neurotransmitters serotonin, melatonin, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline). Most recently identified as cofactor for different substrate formation, NO nitric oxide, also having neurotransmitter function. Enzyme is NOS, nitric oxide synthase.

Health and disease issues PKU, phenylketonuria, excess phenylalanine—PKU can result in severe brain damage, including symptoms of mental retardation, microcenphaly, speech impediments such as stuttering, slurring, and lisps, seizures or convulsions, and behavioral abnormalities. When this condition was first recognized in the 1930's, a significant proportion of all long term patients in mental institutions proved to be undiagnosed phenylketonuriacs. Approximately one person in 45 American whites is a carrier for a defective phenylalanine hydroxylase gene. These mutations are particularly common in people of Celtic origin, but are less frequent in Eastern Europe. Less frequent is THBD, tetrahydrobiopterin deficiency. Rare genetic condition: 450 cases, causing only 1-2% of PKU patients Synthetic tetrahydrobiopterin, developed by BioMarin under the brand name Kuvan and approved by FDA on December 13, 2007, is a synthetic preparation of the dihydrochloride salt of the substance, used in the treatment of PKU Biopterin THB site

Neopterin Biosynthetic precursor in pterin pool; for biopterin cofactor of the aromatic amino acid hydroxylases and nitric oxide synthase. [search Espasy[search Espasy: neopterin] Health and disease issues Induced synthesis from GTP by macrophages upon stimulation with the cytokine interferon-gamma and is indicative of a pro-inflammatory immune status. Neopterin serves as a marker of cellular immune system activation (measured in body fluids: blood serum, cerebrospinal fluid or urine). Why is it formed? A result of activation of GTP cyclohydrolase I; so an excretory product of GTP metabolism. Catabolism to BH4 not induced so neopterin builds up. High neopterin production is associated with increased production of reactive oxygen species, neopterin concentrations also allow to estimate the extent of oxidative stress elicited by the immune system. Increased neopterin production is found in, but not limited to, the following diseases: * viral infections including human immunodeficiency virus (HIV), hepatitis B and Hepatitis C * bacterial infections by intracellular bacteria: borrelia (Lyme Disease) and mycobacterium tuberculosis * parasites such as malaria * autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) * malignant tumor diseases * allograft rejection episodes.

Lower doses of methotrexate have been shown to be very effective for the management of rheumatoid arthritis, Crohn's disease, and psoriasis. For the treatment of rheumatoid arthritis, patients should supplement their diet with folate. In these cases inhibition of dihydrofolate reductase (DHFR) is not thought to be the main mechanism, but rather the inhibition of enzymes involved in purine metabolism, leading to accumulation of adenosine, or the inhibition of T cell activation and suppression of intercellular adhesion molecule expression by T cells. Anti-Folates A number of drugs interfere with the biosynthesis of folic acid and THF. Among them are the dihydrofolate reductase inhibitors such as trimethoprim, pyrimethamine and methotrexate; the sulfonamides (competitive inhibitors of para-aminobenzoic acid in the reactions of dihydropteroate synthetase).