Tetrahydrofolate cobalamins

Metabolism and Mobilization. On entry of vitamin B 2 into the cell, considerable metaboHsm of the vitamin takes place. Co(III)cobalamin is reduced to Co(I)cobalamin, which is either methylated to form methylcobalamin or converted to adenosylcobalamin (coenzyme B>22)- The methylation requires methyl tetrahydrofolate. 

A relatively large number of agents have been utilized to treat this intractable disorder folinic acid (5-formyl-tetrahydrofolic acid), folic acid, methyltetrahydrofolic acid, betaine, methionine, pyridoxine, cobalamin and carnitine. Betaine, which provides methyl groups to the beta i ne ho mocystei ne methyltransferase reaction, is a safe treatment that lowers blood homocysteine and increases methionine. 

This cobalamin-dependent enzyme [EC 2.1.1.13], also known as methionine synthase and tetrahydropteroyl-glutamate methyltransferase, catalyzes the reaction of 5-methyltetrahydrofolate with L-homocysteine to produce tetrahydrofolate and L-methionine. Interestingly, the bacterial enzyme is reported to require 5-adenosyl-L-methionine and FADH2. See also Tetrahydropteroyl-triglutamate Methyltransferase... 

Fig. 1. Folate-cobalamin interaction in the synthesis of purines and pyrimidines and, therefore, of DNA. (1) In gastrointestinal mucosa cells (2) in the liver (3) in peripheral tissues. C, cobalamine DAC, desoxyadenosylcobalamine HC, hydroxy cobalamine MC, methylcobalamine F, folic acid MTHF, methyltetrahydrofolic acid THF, tetrahydrofolic acid DHF, dihydrofolic acid dUMP, deoxyuridinemonophosphate dTMP, deoxythymidine-monophosphate. (Adapted from Far-...

The effects of cobalamin deficiency are most pronounced in rapidy dividing cells, such as the erythropoietic tissue of bone marrow and the mucosal cells of the intestine. Such tissues need both Die N5-N10-methylene and N10-formyl forms of tetrahydrofolate for Ihe synthesis of nucleotides required for DNA replication (see pp. 291, 301). However, in vitamin B12 deficiency, the N5-methyl form of tetrahydrofolate is not efficiently used. Because the methylated fonn cannot be converted directly to other forms of tetrahydrofolate, tie Ns-methyl form accumulates, whereas the levels of the other forms decrease. Thus, cobalamin deficiency is hypothesized to lead to a deficiency of the tetrahydrofolate forms needed in purine and thymine synthesis, resulting in the symptoms of megaloblastic anemia. 

Figure 21-2. Metabolism of homocysteine. BHMT, betaineihomocysteine methyl-transferase CBS, cystathionine P-synthase Cob, cobalamin CTH, cystathionine y-lyase DHF, dihydrofolate DMG, dimethylglycine FAD, flavin adenine dinucleotide MAT, methionine adenosyltransferase 5-MTHF, 5-methyltetrahydrofolate 5,10-MTHF, 5,10-methylenetetrahydrofolate MTHFR, methylenetetrahydrofolate reductase MS, methionine synthase MTRR, methionine synthase reductase MTs, methyl transferases PLE pyridoxal phosphate SAH, S-adenosylhomocysteine SAHH, SAH hydrolase SAM, 5-adenosylmethionine SHMT, serine hydroxymethyltransferase THF, tetrahydrofolate Zn, zinc.

Figure 21-3. The methionine synthase reaction. Methionine synthase catalyzes the remethylation of homocysteine to methionine. In the first half reaction (1), a methyl group is transferred from 5-methyl tetrahydrofolate (5-MTHF) to the reduced form of cobalamin [Cob(I)], generating methyl-cobalamin [Methyl-Cob(III)] and tetrahydrofolate (THF). During the second half reaction (2), the methyl group is transferred from methylcobalamin to homocysteine, generating methionine. During the catalytic reaction, Cob(I) occasionally becomes oxidized, producing an inactive form of cobalamin, cob(II)alamin [Cob(II)]. The enzyme methionine synthase reductase (MTRR) then reactivates Cob(II) through reductive methylation, producing methyl-Cob(III). SAM, 5-adenosylmethionine SAH, 5-adeno-sylhomocysteine.

As shown in Figure 10.9, the overall reaction of methionine synthetase is the transfer of the methyl group from methyl-tetrahydrofolate to homocysteine. However, the enzyme also requires S-adenosyl methionine and a flavoprotein reducing system in addition to the cobalamin prosthetic group. A common polymorphism of methionine synthetase, in which aspartate is replaced by glycine, is associated with elevated plasma homocysteine in some cases, although it is less important than methylene-tetrahydrofolate reductase polymorphisms (Section 10.3.2.1 Harmon etal., 1999). 

Cobalt accepts a methyl group from methyl-tetrahydrofolate, forming methyl Co +-cobalamin. Transfer of the methyl group onto homocysteine results in the formation of Co+-cobalamin, which can accept a methyl group from methyl-tetrahydrofolate to reform methyl Co +-cobalamin. However, except under strictly anaerobic conditions, demethylated Co+-cobalamin is susceptible to oxidation to Co +-cobalamin, which is catalyticaUy inactive. Reactivation of the enzyme requires reductive methylation, with S-adenosyl methionine as the methyl donor, and a flavoprotein linked to NADPH. For this reductive reactivation to occur, the dimethylbenzimidazole group of the coenzyme must be displaced from the cobalt atom by a histidine residue in the enzyme (Ludwig and Matthews, 1997). 

It is interesting that E. coli contains two genes that code for methionine synthase metH for the cobalamin-dependent enzyme and metE for a cobalamin-independent enzyme that depends on an active site Zn + to stabilize deprotonated homocysteine (24). This thiolate species demethylates A -methyl-tetrahydrofolate, which is activated by proton transfer to N-5. MetE is less active ( 100 x ) than MetH, and so in the absence of Bi2 E. coli it produces much more MetE to compensate for the lack of MetH. 

Methylation of homocysteine by 5-methyltetrahydrofolate-homocysteine methyl reductase depends on an adequate supply of 5-methyltetrahydrofoIate. The unmethylated folate is recycled in a cobalamin-dependent pathway, by remethylation to 5,10-methylene-tetrahydrofolate, and subsequent reduction to 5-methyltetrahydrofolate. The transferase enzyme, also named 5,10-methyltretrahydrofolate reductase catalyzes the whole cycle [3,91]. S-adenosylmethionine and 5-methyltetrahydrofolate are the most important methyl unit donors in biological system. S-adenosylmethionine is reported to regulate methylation and transsulfuration pathways in the homocysteine metabolism [3,91]. 

Methylcobalamia is iavolved ia a critically important physiological transformation, namely the methylation of homocysteine (8) to methionine (9) (eq. 2) catalyzed by A/ -methyltetrahydrofolate homocysteine methjitransferase. The reaction sequence involves transfer of a methji group first from A/5 -methjltetrahydrofolate to cobalamin (yielding methjicobalamin) and thence to homocysteine. Once again, the intimate details of the reaction are not weU known (31). Demethylation of tetrahydrofolate to tetrahydrofohc acid is a step in the formation of thymidine phosphate, in turn requited for DNA synthesis. In the absence of the enzyme, excess RNA builds up in ted blood cells. 

The most reduced coenzyme is 5-methyl tetrahydrofolate poly glutamate. It is the source of the methyl group added to homocysteine regenerating methionine and tetrahydrofolate ready to accept a one-carbon unit from formate or serine. This last reaction is where folic acid and vitamin come together (Figs. 8.49, 8.52, and 8.53). The implications of this reaction and how folic acid can mask pernicious anemia are discussed in the seetion on vitamin Big (cyanocobalamin). Note that the formation of 5-methyl-THF nor-mdly is not reversible. Tetrahydrofolate can be regenerated only if there is adequate methyl cobalamin coenzyme. 

Cobalmin Deficiency. Pernicious anemia is the disease associated with vitamin Bi2 deficiency. It is usually caused by the inability to produce intrinsic factor. Indeed, many times the vitamin must be administered by injection. The blood picture, a megaloblastic anemia, is indistinguishable from that caused by folic acid deficiency. Indeed folic acid supplements can mask the blood picture. This is illustrated in Fig. 8.53. Removal of ad-enosyl cobalamin eliminates the regeneration of tetrahydrofolate during the methylation of homocysteine to methionine. Folic acid supplements provide a fresh source of tetrahydrofolate coenzymes. DNA synthesis can continue and new erythrocytes form. Excess folic acid also may compete for the available vitamin, further exacerbating vitamin deficiency. 

Metabolism of cobalamins in mammalian cells. Note the compartmentalization of the synthesis of the two coenzymes adenosylcobalamin (AdoCbl) synthesis occurs in mitochondria, whereas that of methylcobalamin (MeCbl) occurs in cytoplasm. TCII, Transcobalamin II OH-Cbl, hydroxocobalamin T, transport protein for TCII-OHCbl complex FH4, MeFH4, tetrahydrofolate and methyltetrahydrofolate, respectively Cbl, a cobalamin in which the ligand occupying the sixth coordination position of the cobalt is not known. The numerical superscripts adjacent to some of the cobalamins indicate the oxidation state of the cobalt ion. Cobalamins in the +1, +2, and +3 oxidation states are also known as Bn, B 2, and Bi2j, respectively. 

The two reactions in mammalian systems in which cobalamins participate are conversion of L-methylmalonyl-CoA to succinyl-CoA by methylmalonyl-CoA mutase, and methylation of homocysteine to methionine by N -tetrahydrofolate homocysteine methyltransferase (Figure 38-17). The interrelationship of vitamin B12 and folic acid derivatives are discussed below, under Folic Acid. ... 

Methyl trap The sequestering of tetrahydrofolate as N -methyl THF because of decreased conversion of homocysteine to methionine as a result of a deficiency of methionine synthase or its cofactor, cobalamin (vitamin B ). 

Adenosyl-cobalamine catalyzes hydrogen shifts as a special isomerisation reaction. With exception of reduction of ribonucleotides the H-shift occurs intramolecularly. Methyl-cobalamine and tetrahydrofolic add are the coenzymes in methylating homocysteine to methionine. 

Early work in fractionated extracts of both Escherichia coli and liver indicated the participation of a vitamin Bi2-containing protein in the reaction of homocysteine with 5-methyltetrahydrofolate to form methionine and tetrahydrofolate. ° The reaction was stimulated by SAM, although SAM was not the stoichiometric methyl donor. Methylcobalamin was not the primary methyl donor however, it could serve as a methyl donor in the absence of 5-methyltrahydrofolate. It was suggested that methylcobalamin could be a catalytic intermediate in methyl transfer from 5-methyltetrahydrofolate. The role of SAM remained obscure until recent years, when it was found to preserve enzyme activity by maintaining cobalamin as methylcobalamin in the Bi2-protein. 

The conversion of A -methyltetrahydrofolate to methyltetrahydrofolate requires adequate supplies of cobalamin (vitamin Bj ). In vitamin deficiency, A -methyltetrahydrofolate accumulates, whereas supplies of dihydrofolate, tetrahydrofolate, and dTMP are depleted. The answer is (D). 

The primary reaction catalyzed by methionine synthase converts homocysteine (Hey) and methyltetrahydrofolate (CH3H4folate) to methionine and tetrahydrofolate (Figure 2). Occasional oxidation of the reactive cob(I)alamin intermediate produces an inactive cob(II)alamin enzyme, which is reactivated by a reductive methylation that uses S-adenosylmethionine (AdoMet) as the methyl donor and flavodoxin or a flavodoxin-like domain as an electron donor. Thus methionine synthase supports three distinct methyl transfer reactions each involving the cobalamin cofactor. 

Cobalamins Aquocobalamin, methylcobalamin and 5 -deoxyadenosyl-cobalamin (D 10.3) Coenzymes of tetrahydrofolate-dependent methyltransferases and of isomerates (D 10.3), vitamins for humans (vitamin Bj2)... 

Folic acid and cobalamins. Tetrahydrofolic acid is the active form of folic acid and carries Ci compounds such as methanol, formaldehyde, formic acid, etc. In mammals, methionine synthase and methylmalonyl-CoA mutase are the only known enzymes, using methylcobalamin and adenosylcobalamin, respectively, as coenz5unes. 

Folic acid (pteroylglutamic acid) and related compounds are present at high concentration in liver, but spinach, broccoli, peanuts, and fresh fruit are also good dietary sources. The RDA is 300 pg. Folates are important for the synthesis of tetrahydrofolate which is important with cobalamin for a series of 1-carbon transfer reactions leading to DNA synthesis, failure of which leads to megaloblastic anemia. 

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