Homocysteine, and cystathionine

It is apparent that abnormally increased excretion of homocysteine and cystathionine metabolites after a test dose of methionine cannot necessarily be regarded as evidence of vitamin Be deficiency. This means that, like the tryptophan load test, the methionine load test is unreUahle as an index of status in epidemiological studies, although it is (probably) reliable in depletion/ repletion studies to determine requirements. 

From the above work, two unexpected findings emerged. First, crude extracts of spinach also catalyzed a direct reaction of HjS with O-acetylhomoserine to form homocysteine (Giovanelli and Mudd, 1%7). Thus the sulfur moiety of homocysteine could possibly be derived not only from cysteine via transsulfuration, but also directly from HjS via sulfhydra-tion. Second, a variety of O-acylhomoserine esters, but not homoserine itself, were active in the synthesis of both homocysteine and cystathionine (Giovanelli and Mudd, 1966, 1%7) the question arose as to which, if any, of these compounds is of physiological significance as the precursor of the four-carbon portion of homocysteine. 

Methionine is an essential amino acid with a unique role in the initiation of protein synthesis, hi addition, by conversion to 5 -adenosyhnethionine, it serves as the major methyl group donor involved in the formation of creatinine and choline, in the methylation of bases in RNA, and as the source of the aminopropyl group in the formation of polyamines. Finally, in relationship to classical homocystinuria, it is converted by way of homocysteine and cystathionine in a series of reactions termed as the transsulfuration pathway (Fig. 20.3). 

The metabolism of methionine, shown in Figure 11.22, includes two pyridoxal phosphate-dependent steps cystathionine synthetase and cystathionase. Cystathionase activity falls markedly in vitamin deficiency, and as a result there is an increase in the urinary excretion of homocysteine and cystathionine, both after a loading dose of methionine and under basal conditions. However, as discussed below, homocysteine metabolism is affected more by folate status than by vitamin status, and, like the tryptophan load test, the methionine load test is probably not reliable as an index of... 

The most common cause of homocystinuria is a congenital deficiency of cystathionine-p-synthase, a pyridoxine-dependent enzyme that condenses homocysteine and... 

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. 

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.

This pyridoxal-phosphate-dependent enzyme [EC 4.2.99.9], also known as cystathionine y-synthase, catalyzes the reaction of O-succinyl-L-homoserine with L-cysteine to produce cystathionine and succinate. The enzyme can also use hydrogen sulfide and methanethiol as substrates, producing homocysteine and methionine, respectively. In the absence of a thiol, the enzyme can also catalyze a /3,y-elimination reaction to form 2-oxobu-tanoate, succinate, and ammonia. 

The homocystinurias are a group of disorders involving defects in the metabolism of homocysteine. The diseases are inherited as autosomal recessive illnesses, characterized by high plasma and urinary levels of homocysteine and methionine and low levels of cysteine. The most common cause of homocystinuria is a defect in the enzyme cystathionine /3-synthase, which converts homocysteine to cystathionine (Figure 20.21). Individuals who are homozygous for cystathionine [3-synthase deficiency exhibit ectopia lentis (displace ment of the lens of the eye), skeletal abnormalities, premature arte rial disease, osteoporosis, and mental retardation. Patients can be responsive or non-responsive to oral administration of pyridoxine (vitamin B6)—a cofactor of cystathionine [3-synthase. Bg-responsive patients usually have a milder and later onset of clinical symptoms compared with B6-non-responsive patients. Treatment includes restriction of methionine intake and supplementation with vitamins Bg, B, and folate. 

When present in excess methionine is toxic and must be removed. Transamination to the corresponding 2-oxoacid (Fig. 24-16, step c) occurs in both animals and plants. Oxidative decarboxylation of this oxoacid initiates a major catabolic pathway,305 which probably involves (3 oxidation of the resulting acyl-CoA. In bacteria another catabolic reaction of methionine is y-elimination of methanethiol and deamination to 2-oxobutyrate (reaction d, Fig. 24-16 Fig. 14-7).306 Conversion to homocysteine, via the transmethylation pathway, is also a major catabolic route which is especially important because of the toxicity of excess homocysteine. A hereditary deficiency of cystathionine (3-synthase is associated with greatly elevated homocysteine concentrations in blood and urine and often disastrous early cardiovascular disease.299,307 309b About 5-7% of the general population has an increased level of homocysteine and is also at increased risk of artery disease. An adequate intake of vitamin B6 and especially of folic acid, which is needed for recycling of homocysteine to methionine, is helpful. However, if methionine is in excess it must be removed via the previously discussed transsulfuration pathway (Fig. 24-16, steps h and z ).310 The products are cysteine and 2-oxobutyrate. The latter can be oxidatively decarboxylated to propionyl-CoA and further metabolized, or it can be converted into leucine (Fig. 24-17) and cysteine may be converted to glutathione.2993... 

In some organisms sulfur incorporation involves homocysteine as an intermediate. In such cases cysteine formation occurs by a transsulfuration reaction, with the intermediate formation of L,L-cystathionine (fig. 21.86). Cystathionine is formed in a simple condensation reaction from serine and homocysteine by cystathionine-jS synthase. 

The transsulfuration pathway involves conversion of homocysteine to cysteine by the sequential action of two pyridoxal phosphate (vitamin B6)-dependent enzymes, cystathionine- 5-synthase (CBS) and cystathionine y-lyase (Fig. 21-2). Transsulfuration of homocysteine occurs predominantly in the liver, kidney, and gastrointestinal tract. Deficiency of CBS, first described by Carson and Neill in 1962, is inherited in an autosomal recessive pattern. It causes homocystinuria accompanied by severe elevations in blood homocysteine (>100 (iM) and methionine (>60 (iM). Homocystinuria due to deficiency of CBS occurs at a frequency of about 1 in 300,000 worldwide but is more common in some populations such as Ireland, where the frequency is 1 in 65,000. Clinical features include blood clots, heart disease, skeletal deformities, mental retardation, abnormalities of the ocular lens, and fatty infiltration of the fiver. Several different genetic defects in the CBS gene have been found to account for loss of CBS activity. 

Figure 21-1. Structural and metabolic relationships between methionine, homocysteine, and cysteine. CBS, cystathionine b-synthase CTH, cystathionine y-lyase MAT, methionine adenosyltransferase MS, methionine synthase 5-MTHF, 5-methyltetrahydrofoIate MTs, methyl transferases PLR pyridoxal phosphate SAH, S-adenosylhomocysteine SAHH, SAH hydrolase THF, tetrahydrofolate.

CyslAlhionine synthase Biosynthesis of cystathionine from homocysteine and serine... 

Studies of plasma homocysteine and supplements with vitamins that are relevant to enzymes of the pathway of homocysteine breakdown. These enzymes are cystathionine 3-synthase (vitamin Bh), methionine synthase (vitamin Bi ), and 5,10-methylerie-H4folate reductase (folic acid). The associated vitamins are listed,... 

Figure 8 Extended folate metabolism, including compartmentation. MTHFR, methylenetetrahydrofolate reductase SHMT, serine hydroxymethyltransferase BHMT, betaine homocysteine methyltransferase, MAT, methionine adenosyltransferase SAH-hydrolase, S-adenosylhomocysteine hydrolase MT, methyltransferase CBS, cystathionine /i-synthase SAM, S-adenosylmethionine SAH, S-aden-osylhomocysteine THF, tetrahydrofolate and 5-MeTHF, 5-methyltetrahydrofolate. (Reproduced from Van der Put etal. (2001) Folate, homocysteine and neural tube defects An overview. Experimental Biology and Medicine 226 243-270.)...

Methionine, homocysteine, and cysteine are linked by the methylation cycle and transsulfuratlon pathway (Figure 55-9). Conversion of methionine into homocysteine proceeds via the formation of S-adenosyl intermediates including S-adenosylmethionine, die methyl group donor in a wide range of transmethylation reactions. Homocysteine is further condensed with serine by cystathionine 3-synthase to form cystathionine. 

Deficiencies of methionine adenosyltransferase, cystathionine 8-synthase, and cystathionine )/-lyase have been described. The first leads to hypermethioninemia but no other clinical abnormality. The second leads to hypermethioninemia, hyperhomocysteinemia, and homo-cystinuria. The disorder is transmitted as an autosomal recessive trait. Its clinical manifestations may include skeletal abnormalities, mental retardation, ectopia lentis (lens dislocation), malar flush, and susceptibility to arterial and venous thromboembolism. Some patients show reduction in plasma methionine and homocysteine concentrations and in urinary homocysteine excretion after large doses of pyridoxine. Homocystinuria can also result from a deficiency of cobalamin (vitamin B12) or folate metabolism. The third, an autosomal recessive trait, leads to cystathioninuria and no other characteristic clinical abnormality. 

E. Vitamin is a cofactor in two biochemical reactions, the conversion of homocysteine to methionine by the enzyme methionine synthase and the conversion of L-methylmalonyl-CoA to succinyl-CoA by methytmalonyl-CoA mutase. N -methyl THF is a methyl donor in the methionine synthase reaction. A folate deficiency would result in decreased methionine synthase activity and decreases in methionine and cystathionine concentrations, while homocysteine levels would be increased. A vitamin deficiency would also yield these same results, but in addition methytmalonate levels would increase as a consequence of a decrease in the activity of methylmalonyl-CoA mutase activity. 

In /3-replacement reactions, the /3-substituent of an amino acid substrate is replaced by a new /3-substituent. For the three enzymes (TRPS, OASS, and cystathionine /3-synthase (CBS)) whose mechanisms are discussed in this section, the catalytic reaction is composed of two distinct half-reactions. The /3-elimination is followed by a /3-addition where nucleophilic agents, indole, sulfide, and homocysteine, respectively, react with the ci-aminoacrylate Schiff base to form the final product, L-tryptophan, L-cysteine, and L-cystathionine, respectively. 

Cystathionine (made by cystathionine synthase from homocysteine and serine) plays a central role both in the biosynthesis of methionine in plants and bacteria and in the biosynthesis of cysteine in animals. In humans, deficiency of cystathionine synthase leads to a condition called homocystinuria, in which homocysteine overaccumulates. The condition results in severe mental retardation and dislocation of the lens of the eye. 

Treatment is directed toward early reduction of the elevated levels of homocysteine and methionine in the blood. In addition to a diet low in methionine, very high oral doses of pyridoxine (vitamin B6) have significantly decreased the plasma levels of homocysteine and methionine in some patients with cystathionine 3-synthase deficiency. (Genetically determined responders to pyridoxine treatment make up approximately 50% of type I homocystinurics.) PLP serves as a cofactor for cystathionine (3-synthase however, the molecular properties of the defective enzyme that confer the responsiveness to B6 therapy are not known. 

Autotrophic organisms synthesize methionine from asparfafe as shovm in the lower right side of Fig. 24-13. This involves fransfer of a sulfur atom from cysteine info homocysteine, using the carbon skeleton of homoserine, the intermediate cystathionine, and two PLP-dependent enzymes, cystathionine y-synthase and cystathionine p-lyase. This transsulfuration sequence (Fig. 24-13, Eq. 14-33) is essentially irreversible because of the cleavage to pyruvate and NH4+ by the P-lyase. Nevertheless, this transsulfuration pathway operates in reverse in the animal body, which uses two different PLP enzymes, cystathionine P s3mthase (which also contains a bound heme) and cystathionine y-lyase (Figs. 24-13,24-16, steps h and i), in a pathway that metabolizes excess methionine. 

Homocystinuria is a biochemical abnormality caused either by a deficiency of cystathionine P-syn-thase or impaired activity of N -methyltetrahydrofolate-homocysteine methyltransferase. The classical homocystinuria occurs when the conversion of homocysteine to cystathionine is limited by a deficiency of cystathionine P-synthase, with accumulation of methionine and homocysteine and a decrease in cysteine. 

Transmethylation yields 1 mole of adenosine for every mole of methylated product. Patients are known with inborn errors that lead to incomplete metabolism of the homocysteine released after transmethylation. Homocysteinurics release about 3 mg of homocysteine per kilogram on unsupplemented diets, more on supplemented (G3, LI). This is minimal, owing to other fates of homocysteine and possible incompleteness of the block (L2). Cystathioninurics have been reported to excrete over a gram of cystathionine per day and up to 0.6 g per gram of creatinine (F6, H3, K9). Since the latter probably have some cystadiioninase, and some remethylation of homocystine may occur, the reported values are also minimal. Nevertheless, the moles of homocysteine and its products are of the same order as the amount of hypoxanthine formed per day. 

H2S can be produced via the metabolism of sulfhydryl-bearing amino acids, specifically by several enzymes found in the methionine-homocysteine-cysteine pathway such as cystathionine 3 synthase (CBS) and cystathionine lyase (CGL) (Fig. 8.1) [6, 10, 11]. The sequence of CBS has been identified in genomes from bacteria to humans [12-14], and a gene similar to the sulfide quinone oxidoreductase gene has been identified in the genome of flies, worms, mice, rats, and humans [15], indicating that cellular H2S and its regulation may be widespread and essential. 

A number of amino acids act as intermediates in the biosynthesis of various protein amino acids and are thus present in plants, although often in low concentrations. These include homoserine (III), homocysteine (IV), cystathionine (V), ornithine (VI), and citrulline (VII). The properties of these... 

In the nonprotein fraction reduced glutathione, GSH, is ubiquitous, and is commonly a mqjor constituent (Table I). The soluble fraction of plants also includes a variety of other sulfur-containing compounds that are normally present in relatively small amounts (a) Intermediates on the route to protein cysteine and protein methionine, such as cysteine, cystathionine, homocysteine, and methionine, (b) Compounds involved in methyl transfer reactions and polyamine synthesis AdoMet.t AdoHcy, and, presumably, 5 -methyl-thioadenosine. The biochemistry of the compounds in both groups (a) and (b) will be discussed here, (c) Compounds clearly related metabolically to cysteine or methionine, such as 5-methylcysteine and 5-methylmethionine. Because in certain plants these derivatives comprise a major portion of the nonprotein sulfur amino acids, they will be discussed here, (d) A number of compounds of uncertain function, the biochemistry of which has often not been clarified. Discussion of such compounds (Richmond, 1973 Fowden, 1964) is beyond the scope of this chapter. 

---