Pyridoxal-5 -phosphate cystathionine 3-synthase

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. 

This pyridoxal-phosphate-dependent enzyme [EC 4.2.1.22] (also known as serine sulfhydrase, /3-thionase, and methylcysteine synthase) catalyzes the reaction of homocysteine with serine to produce cystathionine and water. 

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. 

Gonversion of homocysteine to Gys occurs in two reactions catalyzed by two pyridoxal phosphate-requiring enzymes, cystathionine p-synthase and y-cystathionase. 

There are two pyridoxal phosphate-requiring enzymes in the homocysteine degradation pathway, which are associated with genetic diseases. In homo-cystinuria, cystathionine synthase is defective, and large amounts of homocystine are excreted in the urine. Some homocystinurics respond to the administration of large doses of vitamin B6. In cystathioninuria, cystathionase is either defective or absent. These patients excrete cystathionine in the urine. Cystathionase is often underactive in the newborns with immature livers, and cysteine and cystine become essential amino acids. Human milk protein is especially rich in cysteine, presumably to prepare the newborn for such a contingency. 

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.

Jhee KH, McPhie P, and Miles EW (2000) Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein. Journal of Biological Chemistry 275,11541. ... 

Kabil O, Toaka S, LoBrutto R, Shoemaker R, and Banerjee R (2001) Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases. Evidence from P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme. Journal of Biological Chemistry 276,19350-5. 

Homocysteine is metabolized in the liver, kidney, small intestine and pancreas also by the transsulfuration pathway [1,3,89]. It is condensed with serine to form cystathione in an irreversible reaction catalyzed by a vitamin B6-dependent enzyme, cystathionine-synthase. Cystathione is hydrolyzed to cysteine that can be incorporated into glutathione or further metabolized to sulfate and taurine [1,3,89]. The transsulfuration pathway enzymes are pyridoxal-5-phosphate dependent [3,91]. This co-enzyme is the active form of pyridoxine. So, either folates, cobalamin, and pyridoxine are essential to keep normal homocysteine metabolism. The former two are coenzymes for the methylation pathway, the last one is coenzyme for the transsulfuration pathway [ 1,3,89,91 ]. 

Figure 7-10. Amino acids that can be converted to succinyl CoA. The amino acids methionine, threonine, isoleucine, and valine, which form succinyl CoA via methylmalonyl CoA, are all essential. The carbons of serine are converted to cysteine and do not form succinyl CoA by this pathway. A defect in cystathionine synthase (M) causes homocystinuria. SAM= S-adenosylmethionine PLP = pyridoxal phosphate.

In homocystinuria, cystathionine synthase is defective. Therefore, homocysteine does not react with serine to form cysteine (see Figure 7-10). The homocysteine that accumulates is oxidized to homocystine and excreted in the urine. Some cases respond to increased doses of vitamin B6, which forms pyridoxal phosphate, the cofactor for the synthase enzyme. 

Kery, V., Bukovska, G., and Kraus, J.P (1994) Transsulfuration depends on heme in addition to pyridoxal 5 -phosphate. Cystathionine beta-synthase is a heme protein. J. Biol. Chem. 269, 25283-25288. 

Cysteine synthesis is a primary component of sulfur metabolism. The carbon skeleton of cysteine is derived from serine (Figure 14.7). In animals the sulfhydryl group is transferred from methionine by way of the intermediate molecule homocysteine. (Plants and some bacteria obtain the sulfhydryl group by reduction of SOj to S2 as H2S. A few organisms use H2S directly from the environment.) Both enzymes involved in the conversion of serine to cysteine (cystathionine synthase and y-cystathionase) require pyridoxal phosphate. 

As noted above, cystathionine formation is the other major fate of methionine. The condensation of homocysteine with serine is catalyzed by the vitamin requiring enzyme cystathionine P-synthase. In the last step of the transsulfuration sequence, cystathionine undergoes cleavage to cysteine and a-ketobutyrate in yet another enzyme reaction that requires pyridoxal phosphate. 

Fig. 20.3 Pathway of methionine metabolism. The numbers represent the following enzymes or sequences (1) methionine adenosyltransferase (2) S-adenosylmethionine-dependent transmethylation reactions (3) glycine methyltransferase (4) S-adenosylhomocysteine hydrolase (5) betaine-homocysteine methyltransferase (6) 5-methyltetrahydrofolate homocysteine methyltransferase (7) serine hydroxymethyltransferase (8) 5,10-methylenetetrahydrofolate reductase (9) S-adenosylmethionine decarboxylase (10) spermidine and spermine synthases (11) methylthio-adenosine phosphorylase (12) conversion of methylthioribose to methionine (13) cystathionine P-synthase (14) cystathionine y-lyase (15) cysteine dioxygenase (16) cysteine suplhinate decarboxylase (17) hypotaurine NAD oxidoreductase (18) cysteine sulphintite a-oxoglutarate aminotransferase (19) sulfine oxidase. MeCbl = methylcobalamin PLP = pyridoxal phosphate...

As the name implies, renal clearance of abnormal levels of homocystine in the plasma causes excessive excretion of the amino acid in the urine. In cystathionine P-synthase deficiency, plasma methionine concentrations are elevated as well -this serves as a point of distinction from the remethylation defects. At present, it appears that the pyridoxal phosphate response may be explained by the fact that this vitamin increases the steady-state concentration of the active enzymes by decreasing the rate of apoenzyme degradation and possibly by increasing the rate of holoenzyme formation. The explanation is not entirely satisfactory, however, since in vitro studies have shown detectable levels of enzyme activity in mutant fibroblasts that have no response, while in other mutant lines without detectable enzyme activity, response has occurred. Once again, a distressing lack of correspondence between in vivo observations and in vitro experiments forces investigators to probe the secrets of these diseases more deeply. 

So, the biosynthesis of methionine (Met, M), the first of the essential amino adds to be considered (Scheme 12.13), begins by the conversion of aspartate (Asp, D) to aspartate semialdehyde in the same way glutamate (Glu, E) was converted to glutamate semialdehyde (vide supra. Scheme 12.6). Phosphorylation on the terminal carboxylate of aspartate (Asp, D) by ATP in the presence of aspartate kinase (EC 2.7.2.4) and subsequent reduction of the aspart-4 yl phosphate by NADPH in the presence of aspartate semialdehyde dehydrogenase (EC 1.2.1.11) yields the aspartate semialdehyde. The aspartate semialdehyde is further reduced to homoserine (homoserine oxoreductase, EC 1.1.1.3) and the latter is succinylated by succinyl-CoA with the liberation of coenzyme A (CoA-SH) in the presence of homoserine O-succinyl-transferase (EC 2.3.1.46). Then, reaction with cysteine (Cys, C) in the presence of cystathionine y-synthase (EC 2.5.1.48) produces cystathionine and succinate. In the presence of the pyridoxal phosphate protein cystathionine P-lyase (EC 4.4.1.8), both ammonia and pyruvate are lost from cystathionine and homocysteine is produced. Finally, methylation on sulfur to generate methionine (Met, M) occurs by the donation of the methyl from 5-methyltetrahydrofolate in the presence of methonine synthase (EC 2.1.1.13). 

MH Lipson, J Kraus, LE Rosenberg. Affinity of cystathionine synthase for pyridoxal 5 -phosphate in cultured cells. J Clin Invest 66 188-193, 1980. 

Cystathionine synthase has pyridoxal phosphate as cofactor a radically different form of treatment was introduced in 1967 giving pyridoxine at a dosage level of 50 to 200 mg per day [40]. The concentrations of methionine and homocysteine in the blood, and of homocystine and homocysteine-cysteine mixed disulphide in the urine, fell sharply on such treatment in... 

Tsai MY, Yang F, Bignell M, Aras O, and Hanson NQ (1999) Relation between plasma homocysteine concentration, the 844ins68 variant ofthe cystathionine beta-synthase gene, and pyridoxal-5 -phosphate concentration. Molecular Genetics and Metabolism 67, 352-6. 

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