Homocysteine cobalamins

Because of the complexity of the enzymatic systems involved in coenzyme Bi2 chemistry there are several reports on the purification of B 12-dependent enzymes or B 12-binding proteins by vitamin B12 affinity adsorbents. In fact, for purification of enzymes or proteins, affinity chomatography has been widely used as one of the most attractive methods (270). For that purpose, the synthesis of a cobalamin-Sepharose insoluble support has been prepared and applied to the purification of iV -methyltetrahydrofolate-homocysteine cobalamin methyltransferase from E. coll The scheme for the synthesis of the solid support is summarized in Fig. 6.14. 

Mechanistic aspects of the action of folate-requiring enzymes involve one-carbon unit transfer at the oxidation level of formaldehyde, formate and methyl (78ACR314, 8OMI2I6OO) and are exemplified in pyrimidine and purine biosynthesis. A more complex mechanism has to be suggested for the methyl transfer from 5-methyl-THF (322) to homocysteine, since this transmethylation reaction is cobalamine-dependent to form methionine in E. coli. 

Enzymatic methylation of homocysteine (HSCHjCHjCHNHjCOOH) by methylcobalamin to give methionine (CH3SCH2CH2CHNH2COOH) was discovered in 1962 by Woods and co-workers, who also noticed the occurrence of a much slower, nonenzymatic reaction giving the same products. Methylcobinamide showed the same activity as the cobalamin in both the enzymatic and nonenzymatic reactions (72, 7/). It was subsequently discovered that HS, MeS , PhS , and w-BuS will dealkylate a variety of methyl complexes [DMG, DMG-BF2, DPG, G, salen, (DO)(DOH)pn, cobalamin] and even ethyl-Co(DMG)2 complexes to give the thioethers, and it was suggested that the reaction involved transfer of the carbonium ion to the attacking thiolate 161, 164), e.g.,... 

In mammals and in the majority of bacteria, cobalamin regulates DNA synthesis indirectly through its effect on a step in folate metabolism, catalyzing the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate via two methyl transfer reactions. This cytoplasmic reaction is catalyzed by methionine synthase (5-methyltetrahydrofolate-homocysteine methyl-transferase), which requires methyl cobalamin (MeCbl) (253), one of the two known coenzyme forms of the complex, as its cofactor. 5 -Deoxyadenosyl cobalamin (AdoCbl) (254), the other coenzyme form of cobalamin, occurs within mitochondria. This compound is a cofactor for the enzyme methylmalonyl-CoA mutase, which is responsible for the conversion of T-methylmalonyl CoA to succinyl CoA. This reaction is involved in the metabolism of odd chain fatty acids via propionic acid, as well as amino acids isoleucine, methionine, threonine, and valine. 

Methionine synthase deficiency (cobalamin-E disease) produces homocystinuria without methylmalonic aciduria 677 Cobalamin-c disease remethylation of homocysteine to methionine also requires an activated form of vitamin B12 677 Hereditary folate malabsorption presents with megaloblastic anemia, seizures and neurological deterioration 678... 

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. 

Cobalamin-c disease remethylation of homocysteine to methionine also requires an activated form of vitamin B12. In the absence of normal B12 activation, homocystinuria results from a failure of normal vitamin B12 metabolism. Complementation analysis classifies defects in vitamin B12 metabolism into three groups cblC (most common), cblD and cblF. Most individuals become ill in the first few months or weeks of life with hypotonia, lethargy and growth failure. Optic atrophy and retinal changes can occur. Methylmalonate excretion is excessive, but less than in methylmalonyl-CoA mutase deficiency, and without ketoaciduria or metabolic acidosis. 

The fibroblasts do not convert cyanocobalamin or hydroxocobalamin to methylcobalamin or adenosyl-cobalamin, resulting in diminished activity of both N5-methyltetrahydrofolate homocysteine methyltransferase and methylmalonyl-CoA mutase. Supplementation with hydroxocobalamin rectifies the aberrant biochemistry. The precise nature of the underlying defect remains obscure. Diagnosis should be suspected in a child with homocystinuria, methylmalonic aciduria, megaloblastic anemia, hypomethioninemia and normal blood levels of folate and vitamin B12. A definitive diagnosis requires demonstration of these abnormalities in fibroblasts. Prenatal diagnosis is possible. 

It is the role of jV5-methyl THF which is key to understanding the involvement of cobalamin in megaloblastic anaemia. The metabolic requirement for N-methyl THF is to maintain a supply of the amino acid methionine, the precursor of S-adenosyl methionine (SAM), which is required for a number of methylation reactions. The transfer of the methyl group from jV5-methyl THF to homocysteine is cobalamin-dependent, so in B12 deficiency states, the production of SAM is reduced. Furthermore, the reaction which brings about the formation of Ns-methyl THF from N5,N10-methylene THF is irreversible and controlled by feedback inhibition by SAM. Thus, if B12 is unavailable, SAM concentration falls and Ah -methyl THF accumulates and THF cannot be re-formed. The accumulation of AT-methyl THF is sometimes referred to as the methyl trap because a functional deficiency of folate is created. 

The vitamin cobalamin (vitamin Bjj) is reduced and activated in the body to two forms, adeno-sylcobalamin, used by methylmalonyl CoA mutase, and methylcobalamin, formed from methyl-THF in the N-methyl THF-homocysteine methyltransferase reaction. These are the only two enzymes that use vitamin (other than the enzymes that reduce and add an adenosyl group to it). 

Cobalamin deficiency can create a secondary deficiency of active THF by preventing its release from the storage pool through the AT-methyl THF-homocysteine methyltransferase reaction, and thus also result in megaloblastic anemia. Progressive peripheral neuropathy also results from cobalamin deficiency. TTeating a cobalamin deficiency with folate corrects the megaloblastic anemia but does not halt the neuropathy. 

Researchers studying the metalloenzyme hydrogenase would like to design small compounds that mimic this enzyme s ability to reversibly reduce protons to H2 and H2 to 2H+, using an active center that contains iron and nickel. Cobalamins (vitamin and its derivatives) contain an easily activated Co-C bond that has a number of biological functions, one of which is as a methyl transferase, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR). This enzyme converts homocysteine (an amino acid that has one more CH2 group in its alkyl side chain than cysteine see Figure 2.2) to methionine as methylcobalamin is converted to cobalamin. 

In animal metabolism, derivatives of cobalamine are mainly involved in rearrangement reactions. For example, they act as coenzymes in the conversion of methylmalonyl-CoA to succinyl-CoA (see p. 166), and in the formation of methionine from homocysteine (see p. 418). In prokaryotes, cobalamine derivatives also play a part in the reduction of ribonucleotides. 

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... 

Cobalamin compounds Tight Cobalamin (B12) Transfer of methyl group to homocysteine during synthesis of methionine metabolism of methylmalonyl coenzyme A... 

Vitamin Bjj (8.50, cobalamin) is an extremely complex molecule consisting of a corrin ring system similar to heme. The central metal atom is cobalt, coordinated with a ribofuranosyl-dimethylbenzimidazole. Vitamin Bjj occurs in liver, but is also produced by many bacteria and is therefore obtained commercially by fermentation. The vitamin is a catalyst for the rearrangement of methylmalonyl-CoA to the succinyl derivative in the degradation of some amino acids and the oxidation of fatty acids with an odd number of carbon atoms. It is also necessary for the methylation of homocysteine to methionine. 

Vitamin B12 Low serum cobalamin (< 150 pmol/L) accompanied by increased serum homocysteine (> 13 Nnol/L), and increased serum (> 0.4 -mol/L) and urine (> 3.6 mmol/mol creatinine) methylmalonic acid... 

Fig. 2.2.1 Outline of homocysteine metabolism in man. BMT Betaine methyltransferase, cblC cobalamin defect type C, cblD cobalamin defect type D, GNMT def glycine N-methyltransferase deficiency, MAT methionine adenosyl transferase, MeCbl methylcobalamin, Met Synth methionine synthase, MTHFR methylenetetrahydrofolate reductase, SAH Hyd dc/S-adenosylhomocys-...

Stabler SP, Marcell PD, Podell ER, Allen RH, Savage DG, Lindenbaum J (1988) Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry. J Clin Invest 81 466-474... 

Vitamin B12 consists of a porphyrin-like ring structure, with an atom of Co chelated at its centre, linked to a nucleotide base, ribose and phosphoric acid (6.34). A number of different groups can be attached to the free ligand site on the cobalt. Cyanocobalamin has -CN at this position and is the commercial and therapeutic form of the vitamin, although the principal dietary forms of B12 are 5 -deoxyadenosylcobalamin (with 5 -deoxyadeno-sine at the R position), methylcobalamin (-CH3) and hydroxocobalamin (-OH). Vitamin B12 acts as a co-factor for methionine synthetase and methylmalonyl CoA mutase. The former enzyme catalyses the transfer of the methyl group of 5-methyl-H4 folate to cobalamin and thence to homocysteine, forming methionine. Methylmalonyl CoA mutase catalyses the conversion of methylmalonyl CoA to succinyl CoA in the mitochondrion. 

Vitamins B6, B12, and folate An elevated plasma homocysteine level is associated with increased cardiovascular risk (see p. 263). Homocysteine, which is thought to be toxic to the vascular endothelium, is converted into harmless amino acids by the action of enzymes that require the B vitamins—folate, B6 (pyridoxine), and B12 (cobalamin). Ingesting foods rich in these vitamins can lower homocysteine levels and possibly decrease the risk of car diovascular disease. Folate and B6 are found in leafy green veg etables, whole grains, some fruits, and fortified breakfast cereals. B12 comes from animal food, for example, meat, fish, and eggs. 

Vitamin B12 Cobalamin Methylcobalamin Deoxyadenosyl cobalamin Cofactor for reactions Homocysteine > Methionine I Methylmalonyl CoA -> Succinyl CoA j... 

Vitamin B12 (cobalamin) has as its active forms, methylcobalamin and deoxyadenosyl cobalamin. It serves as a cofactor for the conversion of homocysteine to methionine, and methylmalonyl CoA to succinyl CoA. A deficiency of cobalamin results in pernicious (megaloblastic) anemia, dementia, and spinal degeneration. The anemia is treated with IM or high oral doses of vitamin B12. There is no known toxicity for this vitamin. 

Hall, M Gamble, M Slavkovich, V. et al. (2007) Determinants of arsenic metabolism Blood arsenic metabolites, plasma folate, cobalamin, and homocysteine concentrations in maternal-newborn pairs. Environmental Health Perspectives, 115 (10), 1503-9. 

Although numerous enzymatic reactions requiring vitamin B12 have been described, and 10 reactions for adenosylcobalamin alone have been identified, only three pathways in man have so far been recognized, one of which has only recently been identified (PI). Two of these require the vitamin in the adenosyl form and the other in the methyl form. These cobalamin coenzymes are formed by a complex reaction sequence which results in the formation of a covalent carbon-cobalt bond between the cobalt nucleus of the vitamin and the methyl or 5 -deoxy-5 -adenosyl ligand, with resulting coenzyme specificity. Adenosylcobalamin is required in the conversion of methylmalonate to succinate (Fig. 2), while methylcobalamin is required by a B12-dependent methionine synthetase that enables the methyl group to be transferred from 5-methyltetrahydrofolate to homocysteine to form methionine (Fig. 3). 

During the remethylation of homocysteine to methionine, the methyl group from 5-MTHF is transferred to cobalamin, which serves as an... 

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.

Measurement of blood tHcy is usually performed for one of three reasons (1) to screen for inborn errors of methionine metabolism (2) as an adjunctive test for cobalamin deficiency (3) to aid in the prediction of cardiovascular risk. Hyperhomocysteinemia, defined as an elevated level of tHcy in blood, can be caused by dietary factors such as a deficiency of B vitamins, genetic abnormalities of enzymes involved in homocysteine metabolism, or kidney disease. All of the major metabolic pathways involved in homocysteine metabolism (the methionine cycle, the transsulfuration pathway, and the folate cycle) are active in the kidney. It is not known, however, whether elevation of plasma tHcy in patients with kidney disease is caused by decreased elimination of homocysteine in the kidneys or by an effect of kidney disease on homocysteine metabolism in other tissues. Additional factors that also influence plasma levels of tHcy include diabetes, age, sex, lifestyle, and thyroid disease (Table 21-1). 

Cobalamin (vitamin B12) Methionine cycle intermediate methyl carrier in the remethylation of homocysteine to methionine cofactor for methionine synthase... 

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