A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e , Vol. 1, No. 8 Copyright © 1971, Institute for Clinical Science The Biochemistry of Folic Acid and Vitamin B12 M. MICHAEL LUBRAN, M.D., P h .D . Harbor General Hospital Torrance, CA 90509 Folic acid and vitamin B12 are the pre­ tissues and human red cells as a polygluta­ cursors of coenzymes involved in many mate, typically containing three to seven important biochemical reactions. Most of glutamic acid residues linked by gamma- our knowledge of the biochemistry of these peptide bonds. Human small intestinal vitamins has been obtained from the study juice contains the enzyme folic conjugase of bacterial metabolism: there have been which is necessary for the hydrolysis and fewer studies of animal or human tissues. absorption of the poly glutamates. Absorp­ The role of these vitamins in man has been tion is an active process that occurs mainly determined mainly by the investigation of in the duodenum and jejunum. patients with folate or BJ2 deficiency. There Folic acid itself has no biological activity. is still much that is not known about their It is the precursor of a group of coenzymes behavior in man; nor is it certain that all derived from tetrahydrofolic acid in which the biochemical reactions in which they the N atoms at 5 and 8 and the C atoms participate have been discovered. Reactions at 6 and 7 of the pyrazine ring have been involving folic acid and B12 in micro-or­ reduced. The tetrahydrofolate derivatives ganisms and even animals do not neces­ contain groups attached to N-5, N-10 or sarily occur in man. In particular, megalo­ both atoms by a bridge (table 1). The blastic anemia as seen in the human does coenzyme is attached to its specific enzyme not occur in animals deficient in folate or through the pyrimidine ring. Bi2. The major biochemical reactions of Folic acid is reduced to dihydrofolic acid folic acid and vitamin Bi2 will be described and then to tetrahydrofolic acid by the here with emphasis on those known or be­ enzyme dihydrofolic acid reductase, which lieved to be important in human metabo­ requires pyridine nucleotides (NADH2 or lism. The literature on the subject is vast. NADPH2). Dihydrofolic acid, which is Recent reviews are cited in the references. the natural substrate, is reduced more readily than folic acid by this enzyme. Folic Acid Folic acid reductases, which convert folic acid to dihydrofolic acid only, have been Folic acid is pteroylglutamic acid (figure described in some bacteria. Their role in 1). It consists of a pteridine portion linked man is uncertain. Dihydrofolic acid reduc­ through p-aminobenzoic acid to L-glutamic tase is widely distributed in bacterial and acid. It is found in plants (its name is de­ animal cells. It is strongly inhibited by rived from the Latin folium, a leaf), animal folic acid antagonists such as aminopterin 236 BIOCHEMISTRY OF FOLIC ACID AND VITAMIN B12 237 Table I. The Tetrahydrofolate H C00H Y '¿;|rCH2-N-<f 'yC0-N-CH-CH2-CH2-C00H Coenzymes Substituent Formula Attached to N Oxidation level pteridine ^-aminobenzoic L-glutamic Methyl c h 3 5 Methanol acid acid Methylene c h 2- 5 and 10 Formaldehyde pteroic acid Methenyl CH = 5 and 10 Formate V---------------------------------------------- y-----------------------------------------------/ Formyl CHO 5 or 10 Formate pteroylglutamic acid (PteGlu) Formimino CH=NH 5 Formate F ig u r e 1. Structural formula of folic acid. Folate coenzymes include tetrahydrofolate and substituted tetrahydrofolates (4-aminopteroylglutamic acid) and metho­ the folate coenzymes are derived from the trexate (4-amino- 10-methylpteroylglutamic following compounds: serine (/3-C) mainly, acid). formate and histidine to a small extent only. Single carbon atoms derived from these Functions of Folic Acid Coenzymes groups are found in: serine (/3-C), formate, The major function of the folate co­ formaldehyde, purines (C-2 and C-8), enzymes is the transfer of one-carbon thymine (5-methyl C) and methionine (5- groups in a variety of synthetic reactions, methyl C). The reactions involved are which depend upon the state of oxidation described below. All are believed to take of the group transferred. The lowest level place in human metabolism. of oxidation is that of methanol (methyl (1) L- serine-glycine interconversion group); next is formaldehyde (methylene Serine hydroxymethyltransferase, tetra­ group); highest is formate (methenyl, hydrofolate and pyridoxal-5-phosphate re- formyl, formimino groups). Coenzymes versibly convert L-serine to glycine. The containing these groups are readily inter­ enzyme is present in human red cells. convertible by specific enzymes, except for CH, OH-CH-COOH + H, PteClu ¡a CH, COOH + 5.10-CH.-H, PteClu 5-methyltetrahydrofolate. The methylene I I NHa NHS level is reduced to methyl by 5, 10- serine tetrahydrofolate glycine methylenetetra- methylenetetrahydrofolate reductase, a hydrofolatc flavo-protein requiring FADH2 (reduced 2) Formimino glutamic acid (FIGlu) con­ flavin adenine dinucleotide); the reverse version reaction does not occur in man. Regenera­ FIGlu is an intermediate in the enzy­ tion of tetrahydrofolate from methyltetra- matic degradation of histidine in animals. hydrofolate is Bj2 dependent and occurs It is converted to glutamic acid by FIGlu during the synthesis of methionine. This formiminotransferase and tetrahydrofolate. process is described in the section on Bi 2. COOH - CH - CH*-CH8 COOH + H4 PteClu ->COOH-CH-CHa-CHa COOH The NADP-dependent enzyme, 5,10-methy- N H N H . I lenetetrahydrofolate dehydrogenase, brings NH = CH formiminoglutamic acid glutamic acid about the interconversion of coenzymes 5-CHNH-iri PtcGlu containing groups at the formaldehyde and formiminotetrahydrofolate formate levels of oxidation. Methenyl and The formiminotetrahydrofolate produced in formyl groups are interconverted by a cyclo- this reaction is converted by cyclodeaminase hydrolase; methenyl and formimino deriva­ into 5,10-methenyltetrahydrofolate; this in tives by a cyclodeaminase. turn is converted by cyclohydrase into 10- The single carbon groups transferred by formyltetrahydrofolate. In patients with 238 LUBBAN folic acid deficiency, the catabolism of Syntheses Involving Folic Acid FIGlu is decreased and its excretion in the Coenzymes urine is increased. Excretion is more marked Folic acid coenzymes are involved in the in the presence of a histidine load. Vitamin synthesis of purines, pyrimidines (and thus, B12 deficiency may also give rise to in­ indirectly, in the synthesis of DNA) and creased urinary excretion of FIGlu, but the methionine. The last synthesis also involves increase is not as marked as in folic acid vitamin B]2 and will be described later. deficiency. 1) Purine synthesis 3) Utilization of formate Folic acid is concerned in the introduc­ Tetrahydrofolate formylase (formate-ac- tion of carbon atoms into positions 8 and 2 tivating enzyme), ATP and tetrahydrofolate in the purine ring; different coenzymes are give rise, reversibly, to 10-formyltetrahy- involved. C-8 is introduced by the forma­ drofolate; this coenzyme transfers the tion of formylglycinamide ribonucleotide formyl group to appropriate substrates (for (FGAR) from glycinamide ribonucleotide example, in the synthesis of purines). (GAR) by 5,10-methenyltetrahydrofolate H* PteGlu + HCOOH + ATP *=» l()-CHO-H4 PteGlu + ADP +Pi formyltetrahydrofolate and GAR transformylase (figure 2). Tetra­ hydrofolate is formed. The formyl group of Only 10-formyltetrahydrofolate participates FGAR later condenses with the amide N in formate transfer in purine synthesis. The to form an imidazole ring. energy of hydrolysis of 5-formyltetrahydro- C-2 is introduced by formylation of 5- folate is too low for this transfer to occur. amino-4-imidazole-carboxamide ribonucleo­ This enzyme can be converted into the 10- tide (AICAR) with 10-formyltetrahydro­ formyl form by ATP and magnesium ions. folate and AICAR transformylase to give 4) Formylation of glutamate 4-formamido-5-imidazoecarboxamide ribo­ This takes place through the action of nucleotide (FAICAR) and tetrahydrofolate glutamate transformylase and 5-formyltetra- (figure 3). AICAR undergoes ring closure hydrofolate; 10-formyltetrahydrofolate is in­ to form inosinic acid, which is subsequently active in this reaction. converted to adenylic, xanthylic and guany- lic acids by appropriate enzyme systems. Glutainate5-CHO-Hi PteGlu formylglutamide -j- II, PteGlu Folate inhibitors (aminopterin, metho­ The reverse reaction is important in for­ trexate) do not inhibit the folate dependent mate metabolism, since formylglutamate is steps of purine synthesis. an intermediate in the conversion of the a-C atom of glycine into active formate. 2) Pyrimidine synthesis Urinary formate excretion rises in folic acid Folic acid is not concerned in the syn­ deficiency. Excretion is increased by oral thesis of the pyrimidine ring, but in the tryptophan but not histidine. introduction of the methyl group of thy- ch2- nh2 ch2- nh I 5,10-CH ■> ' \ * HQ \ H4PteGlu \ +H,PleGlu FICUII 2, ,„tll,j„c. N H N H tion of carbon atom 8 I I into purine ring. Ribose-P Ribose-P GAR FGAR BIOCHEMISTRY OF FOLIC ACID AND VITAMIN B]2 23 9 0 0 II II r HoN C— N * h2nTCvc 2 II ^ IO-CHO-H4PteGlu 8CH ------------ 2------ > 8CH + H4PteGlu / 0 = CH ^ C - N H2N i Ribose-P j Ribose-P F i g u r e 3 . Introduc­ AICAR FAICAR tion of carbon atom 2 into purine ring. 0 CH Ribose-P INOSINIC ACID mine (figure 4) and, in phage infected E. acid are impaired by the antagonists. The coli, the hydroxymethyl group of 5-hy- synthesis of thymidylate is believed to be droxymethylcytosine. The folate coenzyme the rate limiting step in DNA synthesis. is 5,10-methylenetetrahydrofolate. In the synthesis of thymidylate, CH2 is transferred Vitamin Bi2 from the folate coenzyme by thymidylate Vitamin Bi2, although found in most synthetase and simultaneously reduced to animal tissues, is almost exclusively syn­ CH3; concurrently the tetrahydrofolate thesized by micro-organisms, either com­ initially formed is oxidized to dihydrofolate mensals in the animal’s digestive tract or which is then reduced to tetrahydrofolate ingested with animal food.