Cysteine pyridoxal reaction

The amino acid methionine is biosynthesized by a multistep roule that includes reaction of an inline of pyridoxal phosphate (PLP) to give an unsaturated imine. which then reacts with cysteine. What kinds of reactions are occurring in the two steps ... 

Hydrogen sulfide is a well known general metabolite produced on sulfate reduction by certain bacteria. Moreover, organic forms of sulfur can give rise to HS , hence H2S in certain bacteria. Thus, cysteine desulfhydrase (EC 4.4.1.1, cystathionine y-lyase) converts L-cysteine to H2S, pyruvate, and NH3. This enzyme shows a requirement for pyridoxal phosphate and the unstable ami-noacrylic acid is an intermediate (Equation 1) in the reaction ... 

This enzyme [EC 4.4.1.6] catalyzes the pyridoxal-phos-phate-dependent hydrolysis of an 5-alkyl-L-cysteine to generate an alkyl thiol, ammonia, and pyruvate. The reaction is an a,/3-elimination. In yeast, the enzyme may be identical to cystathionine /3-lyase. See also Alliin Lyase... 

This pyridoxal-phosphate-dependent enzyme [EC 4.4.1.10] catalyzes the reaction cysteine with sulfite to produce cysteate and H2S. The enzyme can also catalyze the reaction of two cysteines (thereby producing lanthio-nine) as well as other alkyl thiols as substrates. 

This pyridoxal-phosphate-dependent enzyme [EC 4.4.1.16], also known as selenocysteine reductase, catalyzes the reaction of L-selenocysteine with a reduced acceptor to produce hydrogen selenide, L-alanine, and the acceptor. The enzyme can use dithiothreitol or 2-mercaptoethanol as the reducing agent. Cysteine, serine, or chloroalanine are not alternative substrates for this enzyme. 

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. 

This enzyme [EC 4.1.99.1], also known as L-tryptophan indole-lyase, catalyzes the hydrolysis of L-tryptophan to generate indole, pyruvate, and ammonia. The reaction requires pyridoxal phosphate and potassium ions. The enzyme can also catalyze the synthesis of tryptophan from indole and serine as well as catalyze 2,3-elimination and j8-replacement reactions of some indole-substituted tryptophan analogs of L-cysteine, L-serine, and other 3-substituted amino acids. 

There is a general requirement for pyridoxal-5-phosphate (24, 25, 27, 44) although not all of the activity lost on dialysis is restored by adding the cofactor. This requirement explains the inhibition by hydroxylamine and hydrazine (24, 25). The reaction is a typical pyridoxal-5-phosphate catalyzed a,/ -elimination with a mechanism similar to serine dehydrase and cysteine desulfhydrase (45). The coenzyme is probably bound as a Schiff base with an amino group of the enzyme since there is an absorption maximum at 415 nm in solutions of the purified garlic enzyme (40). The inhibition by L-cysteine is presumably caused by formation of a thiazolidine with the coenzyme (46). Added pyridoxal-5-phosphate also combines directly with the substrate. The dissociation constant for the complex is about 5 X lO M. When this is taken into account, the dissociation constant of the holoenzyme can be shown to be about 5 X 10 M (47). The higher enzyme activity in pyrophosphate buflFer than in Tris or phosphate may be explained by pyrophosphate chelation of metal ions which otherwise form tighter complexes with the substrate and coenzyme (47). This decreases the availability of added coenzyme. 

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 ]. 

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

Cysteine was also an elTective inactivator in vitro] the closely related compound penicillamine (/3,j8-dimethylcysteine) is slightly more reactive - . Penicillamine inhibits the transaminases of rat liver and the glutamic acid decarboxylase of mouse brain . The formation of thiazolidine derivatives by reaction of cysteine or penicillamine with pyridoxal phosphate proceeds... 

Enzymatic decarboxylation169 of L-cysteic acid-35S (equation 86) by the tissues of chicken embryo has been investigated by Fromageot and coworkers170. Enzyme preparations from liver appeared to be the most active, and the authors determined [35S]taurine as well as unreacted 35S-cysteic acid, 35S-/Lsulphonylpyruvic acid and 35S-sulphate. The reaction is inhibited by L-cysteine sulphinic acid, by DL-a-methylcysteic acid, CH2ICOONa, NaCN and by hydroxylamine. The enzyme is activated by pyridoxal phosphate. 

Fig. 39.1. Overview of the synthesis of the nonessential amino acids. The carbons of 10 amino acids may be produced from glucose through intermediates of glycolysis or the TCA cycle. The 11th nonessential amino acid, tyrosine, is synthesized by hydroxylation of the essential amino acid phenylalanine. Only the sulfur of cysteine comes from the essential amino acid methionine its carbons and nitrogen come from serine. Transamination (TA) reactions involve pyridoxal phosphate (PLP) and another amino acid/a-keto acid pair.

Reaction 8, the direct cleavage of L-cystine to produce pyruvate and thiocysteine has been studied in much more detail. An enzyme has been found in a number of Brassica species which cattdyzes the production of pyruvate from L-cystine (Mazelis et al., 1%7). The specificity of this enzyme using six-fold purified B. napobrassica root preparations was limited to L-cystine and 5-methyl-L-cysteine sulfoxide of naturally occurring substrates. The enzyme was completely dependent on added pyridoxal 5 -phosphate. The for L-cystine was 1 vaM and 0.5 pM for pyridoxal phosphate. L-Cysteine was not a substrate but was a competitive inhibitor at low concentrations. This was due to the -SH function since glutathione had the same effect. The Aj for these compounds was 0.15 mM. 

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...

These reactions which lead to homocysteine formation in some creatures and its utilization in others are undoubtedly representative of a general thiol group transfer mechanism. The initial condensation of the donor thiol, most commonly cysteine, with some suitably reactive receptor generates a thioether. The differences in the requirement for O-acylation when starting from serine and homoserine may refiect two completely different mechanisms for this thiol substitution reaction. In the case of serine, the removal of the hydroxyl as hydroxide and the stabilization of an electrophilic centre on the side-chain carbon can be achieved through the pyridoxal phosphate-amino acid adduct. A similar example is in the carbon-carbon condensation between serine and imidazole in tryptophan... 

Ibere is no doubt that such a reaction, catalysed by a pyridoxal phosphate-dependent enzyme, can occur in biological systems. It is quite possible, however, that this only represents a side reaction of other enzymes. Qrstathionase, for example, will act on cystine with the elimination of a cysteine persulphide and pyruvate. The persulphide then reacts with cysteine to eliminate sulphide and re nerate cystine. The complete cycle would constitute a cysteine desulphydrase activity. 

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