Vitamin methionine synthetase

Nitrous oxide exerts a variety of its adverse effects by oxidizing vitamin Bn and rendering it inactive as a coenzyme in many essential metabolic processes. One vitamin dependent enzyme in particular, methionine synthetase, is involved in cell division and is necessary for DNA production. Adverse reproductive and hematologic effects caused by nitrous oxide are thought to be due to inactivation or dysfunction of methionine synthetase resulting in impairment of cell division. 

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. 

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). 

Because vitamin B12 in the oxidized form inactivates methionine synthetase, it could be expected to interfere with folate metabolism because 5-methyltetrahydrofolate would be unable to donate its methyl group. This... 

Homocystinuria may result from one or several abnormalities in the mechanism whereby homocysteine is methylated to form methionine. About half of the patients respond to treatment with pyridoxine and it is thought that the vitamin overcomes a block at the homocysteine/cystathionine level by mass action (C23). However, Schuh et al. (S22) have recently described a patient who responded to vitamin B12. The infant presented with severe developmental delay, homocystinuria, and a megaloblastic anemia. Treatment with cyanocobalamin was without effect but treatment with hydroxocobalamin resulted in a rapid clinical improvement, and the homocystinuria disappeared. Methionine synthetase activity in cell extracts was normal, while cultured fibroblasts showed an absolute growth requirement for methionine. The defect appeared to be limited to methyleobalamin accumulation and an inability to transfer the methyl group from 5-methyltetrahydrofolate to homocysteine. 

This is another rare inherited disorder of vitamin B12 metabolism in which both coenzyme forms, adenosylcobalamin and methylcobalamin, are affected. Methylcobalamin is required for the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine to give methionine. Lack of methylcobalamin results in deficient activity of 2V5-methyltetrahydrofolate-homo-cysteine methyltransferase, resulting in a reduced ability to methylate homocysteine. A failure of methionine synthetase would produce a similar result. 

Vitamin B12 is required by only two enzymes in human metabolism methionine synthetase and L-methylmalonyl-CoA mutase. Methionine synthetase has an absolute requirement for methylcobalamin and catalyzes the conversion of homocysteine to methionine (Fig. 28-5). 5-Methyltetrahydrofolate is converted to tetrahydrofolate (THF) in this reaction. This vitamin B12-catalyzed reaction is the only means by which THF can be regenerated from 5-methyltetrahydrofolate in humans. Therefore, in vitamin B12 deficiency, folic acid can become trapped in the 5-methyltetrahydrofolate form, and THF is then unavailable for conversion to other coenzyme forms required for purine, pyrimidine, and amino acid synthesis (Fig. 28-6). All folate-dependent reactions are impaired in vitamin B12 deficiency, resulting in indistinguishable hematological abnormalities in both folate and vitamin B12 deficiencies. 

Figure 9.5. Methionine load test for vitamin Be status. Methionine synthetase, EC 2.1.1.13 (vitamin Bi2-dependent) 2.1.1.5 (betaine as methyl donor) cystathionine synthetase, EC 4.2.1.22 and cystathionase, EC 4.4.1.1. Relative molecular masses (Mr) methionine, 149.2 homocysteine, 135.2 cystathionine, 222.3 and cysteine, 121.2.

The principal substrate for glutamylation is free tetrahydrofolate one-carbon substituted folates are poor substrates. Because the main circulating folate, and the main form that is taken up into tissues, is methyl-tetrahydrofolate, demethylation by the action of methionine synthetase (Section 10.3.3) is essential for effective metabolic trapping of folate. In vitamin B12 deficiency, when methionine synthetase activity is impaired, there wUl be impairment of the retention of folate in tissues. 

There are two separate homocysteine methyltransferases in most tissues. One uses methyl-tetrahydrofolate as the methyl donor and has vitamin B12 (cohalamin Section 10.8.1) as its prosthetic group. This enzyme is also known as methionine synthetase it is the only homocysteine methyltransferase in the central nervous system. The other enzyme utilizes hetaine (an intermediate in the catabolism of choline Section 14.2.1) as the methyl donor and does not require vitamin B12. 

The Methyl Folate Trap Hypothesis The reduction of meth-ylene-tetrahydrofolate to methyl-tetrahydrofolate is irreversible (Section 10.3.2.1), and the major source of folate for tissues is methyl-tetrahydrofolate. The only metabolic role of methyl-tetrahydrofolate is the methylation of homocysteine to methionine, and this is the only way in which methyl-tetrahydrofolate can be demethylated to yield free tetrahydrofolate in tissues. Methionine synthetase thus provides the link between the physiological functions of folate and vitamin B12. 

Impairment of methionine synthetase activity, for example, in vitamin B12 deficiency or after prolonged exposure to nitrous oxide (Section 10.9.7), will result in the accumulation of methyl-tetrahydrofolate. This can neither be utilized for any other one-carbon transfer reactions nor demethylated to provide free tetrahydrofolate. 

Deficiency of vitamins Bg, B12, or folate are aU associated with elevated plasma homocysteine, with vitamin Bg deficiency as a result of impaired activity of cystathionine synthetase (Section 9.5.5) and folate and vitamin B12 as a result of impaired activity of methionine synthetase (Section 10.3.4). In subjects with apparently adequate intakes of vitamins Bg and B12, supplements of these two vitamins have little or no effect on fasting plasma homocysteine, although additional vitamin Bg reduces the plasma concentration of homocysteine after a test dose of methionine. By contrast, supplements of... 

In mammals, there are only three vitamin B12 -dependent enzymes methionine synthetase, methylmalonyl CoA mutase, and leucine aminomutase. The enzymes use different coenzymes methionine synthetase uses methylcobal-amin, and cobalt undergoes oxidation during the reaction methylmalonyl CoA mutase and leucine aminomutase use adenosylcobalamin and catalyze the formation of a 5 -deoxyadenosyl radical as the catalytic intermediate. 

Methionine synthetase also catalyzes the reduction of nitrous oxide to nitrogen and in so doing generates a hydroxyl radical that results in irreversible inactivation of the enzyme (Frasca et al., 1986). Inactivation of methionine synthetase by nitrous oxide has been used as an acute model of vitamin B12... 

The cause of megaloblastosis is depressed DNA synthesis, as a result of impaired methylation of dCDP to TDP, catalyzed by thymidylate synthetase, but more or less normal synthesis of RNA. As discussed in Section 10.3.3, thymidylate synthetase uses methylene tetrahydrofolate as the methyl donor it is obvious that folic acid deficiency will result in unpaired thymidylate synthesis. It is less easy to see how vitamin B12 deficiency results in impaired thymidylate synthesis without invoking the methyl folate trap hypothesis (Section 10.3.4.1). The main circulating form of folic acid is methyl-tetrahydrofolate before this can be used for other reactions in tissues, it must be demethylated to yield free folic acid. The only reaction that achieves this is the reaction of methionine synthetase (Section 10.8.1). Thus, vitamin B12 deficiency results in a functional deficiency of folate. 

Demyelination is because of failure of the methylation of arginine of myelin basic protein. The nervous system is especially vulnerable to depletion of S-adenosylmethionine in vitamin B12 deficiency because, unlike other tissues, it contains only methionine synthetase, which is vitamin B12-dependent and not vitamin B12-independent homocysteine methyl transferase that uses betaine as the methyl donor (Section 10.3.4 Weir and Scott, 1995). 

Horne DW, Patterson D, and Cook RJ (1989) Effect of nitrous oxide inactivation of vitamin B12-dependent methionine synthetase on the subcellular distribution of folate coenzymes In rat liver. Archives of Biochemistry and Biophysics 270, 729-33. 

One of the biochemical adverse effects of nitric oxide is inactivation of vitamin B12, with subsequent potentiation of folate deficiency (19). This effect is mediated by irreversible oxidation of the cobalt residue in vitamin B12 to its Co++ and Co forms. This leads to a reduction in methionine synthetase activity, with downstream effects on DNA synthesis. Previous studies have identified five patients with unsuspected vitamin B12 deficiency who developed subacute combined degeneration of the spinal cord following inhalation anesthesia with nitrous oxide... 

Nitrous oxide inactivates the enzyme methionine synthetase, and caution is urged in giving nitrous oxide to patients who may be deficient in vitamin B12. Low serum vitamin B12 concentrations have previously been reported in patients with sickle cell disease, but the reason for this is uncertain. Three cases of peripheral neuropathy have been reported in patients with sickle cell disease who received nitrous oxide (12-14). AU three had a history of frequent painful sickle crises, for which they received nitrous oxide for prolonged periods. Serum vitamin B12 concentrations were slightly reduced in two patients and very low in the third. The patients aU presented with difficulty in walking and paresthesia. Peripheral sensorimotor neuropathy was confirmed by nerve conduction studies. The patients all responded well to vitamin B12 injections and avoiding further exposure to nitrous oxide. Caution is therefore recommended when using nitrous oxide in patients with sickle cell disease or who are suspected of vitamin B12 deficiency. Two cases of polyneuropathy have also been reported after the use of nitrous oxide for 80 minutes and 3 hours in patients who were subsequently found to have pernicious anemia. They both responded well to hydroxocobalamin. 

---