Methionine synthetase

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. 

In addition, vitamin B12 has a role in the metabolism of cyanide, forming cyanocobalamin. This prevents the binding of cyanide to cytochrome oxidase and permits (relatively slow) metabolism to yield thiocyanate. 

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). 

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 

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There appear also to be toxic effects. In animals, nitrous oxide has been shown to inactivate methionine synthetase which prevents the conversion of deoxyuridine to thymidine and thus has the potential for inducing megaloblastic anemia, leukopenia, and teratogenicity (44—46). A variety of epidemiologic surveys suggest positive correlations between exposure to nitrous oxide and spontaneous abortion in dental assistants (47). 

In this reaction sequence acetic acid synthesis requires methyl transfer as CH3 to a Co(I)-corrin by N2 5-methyltetrahydrofolate monoglutamate to give a methylcorrinoid intermediate which is carboxylated to give a carboxymethylcorrinoid. This carboxymethylcorrinoid would then be reductively removed by NADPH to give acetic acid and regenerate the Co(I)-corrin. In contrast to the methyl-transfer proposed for the methionine synthetase reaction, this mechanism suggests that CH3-stabilized by Co attacks CO2 to give a carboxymethylcorrinoid intermediate. 

Scheme 1 The ethylene biosynthetic pathway. The enzymes catalyzing each step are shown above the arrows. SAM S-adenosyl-L-methionine SAMS S-adenosyl-i-methionine synthetase ACC 1-aminocyclopropane-1-carboxylic acid ACS 1-aminocyclopropane-1-carboxylate synthase ACO 1-aminocyclopropane-1-carboxylate oxidase Ade adenine MTA methylthioadenosine. The atoms of SAM recycled to methionine through methionine cycle are marked in red and the atoms of methionine converted to ethylene are marked in bold. For details see text.

This enzyme [EC 2.5.1.6], also known as 5 -adenosyl-methionine synthetase, catalyzes the reaction of ATP... 

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. 

The biochemistry of coenzyme B12 generally revolves around either mutase enzyme activity, involving functional group migration, notably by stereospecific 1,2-shifts (Scheme 2.8), or methylation by methionine synthetase. The general mechanism for the mutase activity is a radical-based one and has been established by EPR spectroscopy to be of the general form shown in Scheme 2.9. 

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). 

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. 

Secondly, adenosylmethionine synthetase, in addition to synthetase reaction, catalyses tripolyphosphatase reactions stimulated by adenosylmethionine. Both of the enzymatic activities of the enzyme, which has been purified to homogeneity from E. coli, require a divalent metal ion and are markedly stimulated by certain monovalent cations (Markham et al, 1980). Tripolyphosphatase activity is also associated with S-ade nosy I methionine synthetase isozymes from rat liver (Shimizu et al, 1986). 

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. 

The enzyme also known as methionine synthetase catalyzes the conversion of homocysteine to methionine using a folate derivative as the methyl donor. The assay of Garras et al. (1991) is based on quantitation of the o-phthaldehyde derivative of methionine. 

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. 

Methylene-Tetrahydrofolate Reductase The reduction of methylene-tetrahydrofolate to methyl-tetrahydrofolate, shown in Figure 10.7, is catalyzed hy methylene-tetrahydrofolate reductase, a flavin adenine dinucleotide-dependent enzyme during the reaction, the pteridine ring of the substrate is oxidized to dihydrofolate, then reduced to tetrahydrofolate by the flavin, which is reduced by nicotinamide adenine dinucleotide phosphate (NADPH Matthews and Daubner, 1982). The reaction is irreversible under physiological conditions, and methyl-tetrahydrofolate - which is the main form of folate taken up into tissues (Section 10.2.2) - can only be utilized after demethylation catalyzed by methionine synthetase (Section 10.3.4). 

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. 

Unlike most enzymes utilizing or metabolizing tetrahydrofolate, methionine synthetase has equal activity toward methyl-tetrahydrofolate mono- and polyglutamates. As discussed in Section 10.2.2, demethylation of methyl-tetrahydrofolate is essendal for the polyglutamylation and intracellular accumulation of folate. 

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... 

The cobalt atom is in the Co + oxidation state in hydroxo-, aquo-, methyl-, and cyanocobalamins in the Co+ oxidation state in adenosylcobalamin and, transiently, in the demethylated prosthetic group of methionine synthetase (Section 10.8.1). 

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). 

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. 

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