Methionine demethylation

S-adenosyl-L-methionine (AdoMet, SAM) is a cofactor and the most important donor of the methyl (CH3-) group for methyltransferases, including COMT. When the methyl-group has been transferred, the remaining demethylated compound is called S-adenosyl-L-homo-cysteine. 

The biological role of PIMT involves the selective methylation of isoaspartate residues followed by a demethylation step to reform the succi-nimide intermediate. The demethylation causes the release of methanol which can be converted to formaldehyde and finally to formic acid, as demonstrated in rat brain preparations. It was found that S-adenosyl-methionine (SAM), the methyl donor, caused formaldehyde levels to rise in the rat brain homogenates, thus suggesting that excessive formaldehyde may be a precipitating factor in Parkinsons s disease (PD) (Lee et ah, 2008). It is possible that carnosine could suppress formaldehyde toxicity by reacting with it to generate a carnosine-formaldehyde adduct. This should be a relatively easy experiment to perform to test this prediction. 

Homocysteine. A sulfur-containing amino acid, a homologue of cysteine, produced by the demethylation of methionine, and an intermediate in the biosynthesis from methionine via cystathionine. 

Plants and bacteria produce the reduced sulfur required for the synthesis of cysteine (and methionine, described later) from environmental sulfates the pathway is shown on the right side of Figure 22-13. Sulfate is activated in two steps to produce 3-phosphoadeno-sine 5 -phosphosulfate (PAPS), which undergoes an eight-electron reduction to sulfide. The sulfide is then used in formation of cysteine from serine in a two-step pathway. Mammals synthesize cysteine from two amino acids methionine furnishes the sulfur atom and serine furnishes the carbon skeleton. Methionine is first converted to 5-adenosylmethionine (see Fig. 18-18), which can lose its methyl group to any of a number of acceptors to form A-adenosylhomocysteine (adoHcy). This demethylated product is hydrolyzed to free homocys-... 

One recent related study has been reported, tying together isomescaline and schizophrenia. Through the use of radioactive labelling, the extent of de-methylation (the metabolic removal of the methyl groups from the methoxyls) was determined in both schizophrenic patients and normal subjects. When there was a loading of the person with methionine (an amino acid that is the principal source of the body s methyl groups), the schizophrenics appeared to show a lesser amount of demethylation. 

This observation was explained by the assumption that a portion of the protoberberine was formed via norreticuline (96) present in the same incubation mixture and derived from enzymic demethylation of reticuline. Reaction of 96 with an unlabeled one-carbon fragment and subsequent ring closure would then lead to C-8 unlabeled protoberberines. The authors suggest that this one-carbon fragment may be derived from S-adenosyl-methionine, and that the product of its combination with 96 may be converted directly to 91 or 94 without the intermediacy of free reticuline (99). If their assumption is correct, the conversion of norreticuline to the protoberberine alkaloids may not involve the formation of reticuline itself, a suggestion that is at variance with the known intermediacy of reticuline in the biosynthesis of alkaloids of this group. 

FAHN, W LAUSSERMAIR, E DEUS-NEUMANN, B., STOCKIGT, J, Late enzymes in vindoline biosynthesis. S-adenosyl-L-methionine ll-0-demethyl-17-O-deacetylvindoline 11-0-methyltransferase and an unspecific acetylesterase. Plant Cell Rep., 1985, 4,337-340. 

FAHN, W LAUSSERMAIR, E., NEUMANN, B. D., STOCKIGT, J., Late enzymes of vindoline biosynthesis - S-Adenosyl-L- Methionine-1 l-O-Demethyl-17-... 

Strong protic acids cleave phenolic methyl ethers. Thus hydrogen bromide accomplished the same double demethylation as that depicted in Scheme 4.104.189 The final step in a Merck synthesis of the potent dopamine agonist (A,R)-4-pro-pyl-9-hydroxy naphthoxazine (106 2), a phenolic ether deprotection was accomplished on a large scale using methanesulfonic acid in the presence of methionine as the nucleophile [Scheme 4.106]. 190 191... 

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). 

As shown in Figure 10.9, the methyl donor is S-adenosyl methionine, which is demethylated to S-adenosyl homocysteine. After removal of the adenosyl group, homocysteine may undergo one of two metabolic fates remethylation to methionine or condensation with serine to form cystathionine, foUowed by cleavage to yield cysteine - the transulfuration pathway (Section 9.5.5). Cystathionine synthetase has a relatively low Tni compared with normal intra-ceUular concentrations of homocysteine. It functions at a relatively constant rate, and under normal conditions, most homocysteine wUl be remethylated to methionine. 

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. 

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). 

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). 

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. 

The key intermediate in the catalytic pathway is the supemucleophile cob(l)alamin, which attacks A -methyl-tetrahydrofolate, generating tetrahydrofolate and MeCbl. Then homocysteine (probably as its thiolate) attacks MeCbl, which yields methionine and regenerates cob(l)alamin (Scheme 2). The demethylation of A -methyltetrahydrofolate is not trivial, even for the supemucleophilic cob(l)alamin, and considerable efforts have been invested into understanding this reaction, dubbed improbable by Duilio Arigoni. The obvious mode of activation is by proton transfer to N-5 of A -methyl tetrahydrofolate, but as this is weakly basic (pAa 5.1) the nature of the proton source and mode of transfer has been difficult to pin down. Recent research from the Matthews group has shown how the reactivities of cob(I)alamin and methylcobalamin are modulated by the ligand trans to the lone pair of cob(l)alamin and methyl group of methylcobalamin (21). 

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. 

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. 

---