Homocysteine reactions

Phosphohomoserine is used as a substrate for synthesis of cystathionine [Reaction (4)J and homocysteine [Reaction (5)J by extracts of all plants examined (Datko et al., 1974, 1977), but not by any microorganism tested (See Giovanelli et al., in press). Plants are therefore unique among the organisms studied in using 0-phosphohomoserine for cystathionine or homocysteine synthesis. 

Methylcobalamin is involved in a critically important physiological transformation, namely the methylation of homocysteine (8) to methionine (9) (eq. 2) catalyzed by A/ -methyltetrahydrofolate homocysteine methyltransferase. The reaction sequence involves transfer of a methyl group first from... 

The antiviral activity of (5)-DHPA in vivo was assessed in mice inoculated intranasaHy with vesicular stomatitis vims ( 5)-DHPA significantly increased survival from the infection. (5)-DHPA did not significantly reduce DNA, RNA, or protein synthesis and is not a substrate for adenosine deaminase of either bacterial or mammalian origin. However, (5)-DHPA strongly inhibits deamination of adenosine and ara-A by adenosine deaminase. Its mode of action may be inhibition of Vadenosyl-L-homocysteine hydrolase (61). Inhibition of SAH hydrolase results in the accumulation of SAH, which is a product inhibitor of Vadenosylmethionine-dependent methylation reactions. Such methylations are required for the maturation of vital mRNA, and hence inhibitors of SAH hydrolase may be expected to block vims repHcation by interference with viral mRNA methylation. 

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. 

N5-Methyltetrahydrofolate homocysteine methyl-transferase (= methionine synthase). This reaction is essential to restore tetrahydrofolate from N5-methyltetrahydrofolate (Fig. 2). 

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. 

Figure 12.2 Adenosine metabolism. Intracellular adenosine concentrations depend on the balance between energy storage and breakdown. The most important enzymes catalyzing the reactions are indicated. SAH, S-adenosyl-homocysteine ENTs equilibrative nucleoside transporters CNTs, concentrating nucleoside transporters.

Thiolation of peptides and other small molecules containing amines proceeds easily with N-acetyl homocysteine thiolactone. However, protein modification often results in much lower yields unless the reaction is done for extended periods at pH 10-11. 

Add N-acetyl homocysteine thiolactone (Aldrich) to the bicarbonate reaction mixture to obtain a concentration representing a 10- to 20-fold excess over the amount of amines present. For protein thiolation, add the same molar excess of thiolactone reagent to the water reaction medium, and then slowly add an equivalent molar quantity of silver nitrate (AgNO j). Maintain the pH at 7.0-7.5 with periodic addition of NaOH. 

Remove unreacted N-acetyl homocysteine thiolactone and reaction by-products by gel filtration or dialysis against lOmM sodium phosphate, 0.15M NaCl, lOmM EDTA, pH 7.2. Other buffers suitable for individual protein stability may be used as desired. For the silver nitrate-containing reaction, removal of the silver-thiourea complex may be done by adsorption onto Dowex 50, and the protein subsequently eluted from the resin by 1M thiourea. Removal of the thiourea then may be done by gel filtration or dialysis. 

Transfer of a methyl group from S-adenosylmethionine yields S-adenosylhomocysteine, which potently inhibits several methyltransferases this may partially explain the pathology of homocystinuria. Tissue levels of S-adenosylhomocysteine ordinarily are very low, since this metabolite is rapidly cleaved by a specific hydrolase to homocysteine and adenosine (Fig. 40-4 reaction 3). 

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

One form of remethylation deficit involves defective metabolism of folic acid, a key cofactor in the conversion of homocysteine to methionine. Methylenetetra-hydrofolate reductase (Fig. 40-4 reaction 11) reduces... 

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. 

Methionine synthase deficiency (cobalamin-E disease) produces homocystinuria without methylmalonic aciduria. This enzyme mediates the transfer of a methyl group from methyltetrahydrofolate to homocysteine to yield methionine (Fig. 40-4 reaction 4). A cobalamin group bound to the enzyme is converted to methylcobalamin prior to formation of methionine. 

Intramolecular replacement of sulfur by nitrogen has been reported in the complex of [Pt(dien)]2+ with S-guanosyl-L-homocysteine (sgh) according to reaction 2,... 

SMM synthesis is mediated by the enzyme methionine S-methyltransferase (MMT) through the essentially irreversible, AdoMet-mediated methylation of methionine.48"5 Both MMT and SMM are unique to plants 48,50 The opposite reaction, in which SMM is used to methylate homocysteine to yield two molecules of methionine, is catalyzed by the enzyme homocysteine S-methyltransferase (HMT).48 Unlike MMT, HMTs also occur in bacteria, yeast, and mammals, enabling them to catabolize SMM of plant origin, and providing an alternative to the methionine synthase reaction as a means to methylate homocysteine. Plant MMT and HMT reactions, together with those catalyzed by AdoMet synthetase and AdoHcy hydrolase, constitute the SMM cycle (Fig. 2.4).4... 

However, the reaction of NP with thiols may be a necessary but not sufficient cause for the release of NO from the ion as there are many thiols in frog heart tissue and NP is a vasodilator only under illumination. Furthermore Sogo et al. [50] could not detect NO generation from NP in human plasma containing cysteine, glutathione, homocysteine and reduced cysteine residues. Therefore, there must be a unique component of mammalian tissues which is involved in the release of NO from NP, and this reaction comes after reaction with thiol. Kowaluk et al. [51] report that NP is readily metabolised to NO in subcellular fractions of bovine coronary arterial smooth muscle and that the dominant site of metabolism is in the membrane fraction. This led to the isolation of a small membrane-bound protein or enzyme that can convert NP into NO. The mechanism shown in Scheme 8.2 combines the thiol reaction and that with an enzyme. 

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. 

Fig. 7.11. Metabolism of S-(l- [(2,3,4,5-tetrahydro-2-oxothiophen-3-yl)amino]carbonyl ethyl) thiophene-2-carbothioate (MR 889, 7.71) in rats to the active thiol metabolite homocysteine thiolactone thiolactamide (7.72) and to thiophene-2-carboxylic acid (7.73) [154], Subsequent reactions of hydrolysis and conjugation are also shown.

Important pathways requiring SAM include synthesis of epinephrine and of the 7-methylgua-nine cap on eukaryotic mRNA, Synthesis of SAM from methionine is shown in Figure T17-3. After donating the methyl group, SAM is converted to homocysteine and remethylated in a reaction catalyzed by N-methyl THF-homocysteine methyltransferase requirii both vitamin Bj2 and N-meth d-THF. The methionine produced is once again used to make SAM. 

Additional folate may be stored as the highly reduced JV -methyl-THF. This form is referred to as the storage pool as there is only one known enzyme that uses it, and in turn moves it back into the active pool. This enzyme is N-methyl THF-homocysteine methyltransferase, discussed above, which also requires vitamin and is involved in regenerating SAM as a methyl donor for reactions. 

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. 

A high level of homocysteine is an indication of a low rate of conversion of homocysteine to methionine and hence a low level of the methylating agent S-adenosyl methionine. The latter, plus the toxic effects of homocysteine, provides a link between the reactions listed above and three diseases cardiovascular disease (Chapter 22), senile dementia (Chapter 14) and cancer (Chapter 21). 

Figure 15.5 Four reactions involved in methylatlon. The reactions are (1) formation of S-adenosylmethlonIne (SAM) (11) transfer of methyl group to an acceptor (111) conversion of S-adenosylmethlonIne to homocysteine (Iv) conversion of homocysteine to methionine using methyl tetrahydrofolate as the methyl donor with the formation of FH4.

Figure 22.7 Homocysteine formation from methionine and formation of thiolactone from homocysteine. The homocysteine concentration depends upon a balance between the activities of homocysteine methyltransferase (methionine synthase) and cystathionine p-synthase. Both these enzymes require vitamin B12, so a deficiency can lead to an increase in the plasma level of homocysteine. (For details of these reactions, see Chapter 15.) Homocysteine oxidises spontaneously to form thiolactone, which can damage cell membrane.

A simple observation led to the identification of homocysteine as a risk factor for coronary heart disease. Homocysteine is an intermediate in metabolism of the amino acid methionine. Indeed, the first reaction in the catabolism of methionine involves the formation of homocysteine but it can be converted back to methionine in a reaction that is catalysed by methionine synthase (see Figure 22.7). 

In methylcobalamin, X is a methyl group. This compound functions as a coenzyme for several methyltransferases, and among other things is involved in the synthesis of methionine from homocysteine (see p. 418). However, in human metabolism, in which methionine is an essential amino acid, this reaction does not occur. 

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