Pyridoxal phosphate tryptophan synthesis

By contrast, the cytoplasmic decarboxylation of dopa to dopamine by the enzyme dopa decarboxylase is about 100 times more rapid (Am 4x 10 " M) than its synthesis and indeed it is difficult to detect endogenous dopa in the CNS. This enzyme, which requires pyridoxal phosphate (vitamin B6) as co-factor, can decarboxylate other amino acids (e.g. tryptophan and tyrosine) and in view of its low substrate specificity is known as a general L-aromatic amino-acid decarboxylase. 

Following the synthesis of 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase, the enzyme aromatic amino acid decarboxylase (also known as 5-HTP or dopa decarboxylase) then decarboxylates the amino acid to 5-HT. L-Aromatic amino acid decarboxylase is approximately 60% bound in the nerve terminal and requires pyridoxal phosphate as an essential enzyme. 

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. 

Pyridoxal phosphate is the coenzyme for the enzymic processes of transamination, racemization and decarboxylation of amino-acids, and for several other processes, such as the dehydration of serine and the synthesis of tryptophan that involve amino-acids (Braunstein, 1960). Pyridoxal itself is one of the three active forms of vitamin B6 (Rosenberg, 1945), and its biochemistry was established by 1939, in considerable part by the work of A. E. Braunstein and coworkers in Moscow (Braunstein and Kritzmann, 1947a,b,c Konikova et al 1947). Further, the requirement for the coenzyme by many of the enzymes of amino-acid metabolism had been confirmed by 1945. In addition, at that time, E. E. Snell demonstrated a model reaction (1) for transamination between pyridoxal [1] and glutamic acid, work which certainly carried with it the implication of mechanism (Snell, 1945). 

Vitamin B6 occurs naturally in three related forms pyridoxine (6.26 the alcohol form), pyridoxal (6.27 aldehyde) and pyridoxamine (6.28 amine). All are structurally related to pyridine. The active co-enzyme form of this vitamin is pyridoxal phosphate (PLP 6.29), which is a co-factor for transaminases which catalyse the transfer of amino groups (6.29). PLP is also important for amino acid decarboxylases and functions in the metabolism of glycogen and the synthesis of sphingolipids in the nervous system. In addition, PLP is involved in the formation of niacin from tryptophan (section 6.3.3) and in the initial synthesis of haem. 

In vitamin Be-deflcient experimental animals, there are skin lesions (e.g., acrodynia in the rat) and fissures or ulceration at the corners of the mouth and over the tongue, as well as a number of endocrine abnormalities defects in the metabolism of tryptophan (Section 9.5.4), methionine (Section 9.5.5), and other amino acids hypochromic microcytic anemia (the first step of heme biosynthesis is pyridoxal phosphate dependent) changes in leukocyte count and activity a tendency to epileptiform convulsions and peripheral nervous system damage resulting in ataxia and sensory neuropathy. There is also impairment of immune responses, as a result of reduced activity of serine hydroxymethyltransferase and hence reduced availability of one-carbon substituted folate for nucleic acid synthesis (Section 10.3.3). It has been suggested... 

Pyridoxal phosphate is the coenzyme in a large number of amino acid reactions. At this point it is convenient to consider together 1,he mechanism of those pyridoxal-dependent reactions concerned with aromatic amino acids. The reactions concerned are (1) keto acid formation (e.g., from kynurenine, above), 2) decarboxylation (e.g., of 5-hydroxytrypto-phan to 5-hydroxytryptamine, p. 106), (3) scission of the side claain (e.g., 3-tyrosinase, p. 78 tryptophanase, p. 110 and kynureninase, above), and 4) synthesis (e.g., of tryptophan from indole and serine, p. 40). Many workers have considered the mechanism of one or more of these reactions (e.g., 24, 216, 361, 595), but a unified theory is primarily due to Snell and his colleagues (summarized in 593). Snell s experiments have been carried out largely in vitro, and it should be emphasized that in vivo it is the enzyme protein which probably directs the electromeric changes. 

Tryptophan is an essential amino acid involved in synthesis of several important compounds. Nicotinic acid (amide), a vitamin required in the synthesis of NAD+ and NADP+, can be synthesized from tryptophan (Figure 17-24). About 60 mg of tryptophan can give rise to 1 mg of nicotinamide. The synthesis begins with conversion of tryptophan to N-formylkynurenine by tryptophan pyrrolase, an inducible iron-porphyrin enzyme of liver. N-Formylkynurenine is converted to kynurenine by removal of formate, which enters the one-carbon pool. Kynurenine is hydroxylated to 3-hydroxykynurenine, which is converted to 3-hydroxyanthranilate, catalyzed by kynureninase, a pyridoxal phosphate-dependent enzyme. 3-Hydroxyanthranilate is then converted by a series of reactions to nicotinamide ribotide, the immedi-... 

The spectroscopic characteristics of many enzymes and substrates change on formation of an ES complex. These changes are particularly striking if the enzyme contains a colored prosthetic group. Tryptophan synthetase, a bacterial enzyme that contains a pyridoxal phosphate (PLP) prosthetic group, provides a nice illustration. This enzyme catalyzes the synthesis of I.-tryptophan from L-serine and an indole derivative. The addition of L-serine to the enzyme produces a marked increase in the fluorescence... 

The quaternary nitrogen acts as an electron sink, which facilitates the decarboxylation. Further electron and proton shifts produce a Schiff base between the amine and pyridoxal phosphate, which is then hydrolyzed. Amino acid decarboxylations are involved in the synthesis of several metabolically important amines, e.g., 5-hydroxytryptamine (serotonin) from tryptophan, histamine from histidine, and y-aminobutyric acid (GABA) from glutamate. 

NB Pyridoxal phosphate deficiency also compromises the synthesis of NAD+ etc. from tryptophan (see niacin. Chapter 53)... 

Pyridoxal phosphate has been established as a coenzyme in two reactions involving tryptophan. An enzyme has been isolated from Neurospora which catalyzes a synthesis of tryptophane from serine and indole. This reaction requires pyridoxal phosphate. An enzyme has been isolated from E. coli which causes the decomposition of tryptophan to pyruvic acid, indole, and ammonia here too pyridoxal phosphate is a necessary cofactor. 

Synthesis of Tryptophan from Indole and Serine, Tatum and Bonner reported that a mutant of Neurospora crassa could utilize indole in place of tryptophan. Umbreit, Wood, and Gunsalus, by using extracts from this mutant, showed that, with pyridoxal phosphate as a coenzyme, tryptophan was synthesized from serine and indole via the following reaction ... 

Kynureninase is a pyridoxal phosphate (vitamin Bg)-dependent enzyme, and its activity is extremely sensitive to vitamin depletion. Indeed, the ability to metabolize a test dose of tryptophan has been used to assess vitamin B nutritional status (section 11.9.5.1). Deficiency of vitamin B will lead to severe impairment of NAD synthesis from tryptophan. Kynureninase is also inhibited by oestrogen metabolites. 

In certain bacteria there is a specific nutritional requirement for D-amino acids which are found as components of cell structures or antimetabolites. Bacteria normally meet this need by the conversion of L-amino acids to D-amino acids and in the case of alanine, methionine and tryptophan the evidence suggests that these reactions are directly catalysed by amino acid racemases which have a cofactor requirement for pyridoxal phosphate . An oxidation-reduction cofactor may also be a general feature of racemases of this class. However, the mode of epimerisation of L-phenylalanine to D-phenylalanine necessary for the synthesis of some peptide antibiotics, proceeds in an entirely different way, which as yet has only been partially resolved. 

Again, this does not seem to be due to correction of vitamin Bg deficiency, but rather to a direct effect of pyridoxal phosphate on the metabolism of tryptophan. High concentrations of pyridoxal phosphate attenuate the response to glucocorticoid hormones tryptophan dioxygenase is a glucocorticoid-induced enzyme, and thus its synthesis and activity will be reduced by high intakes of vitamin Bg. This reduces the oxidative metabolism of tryptophan and increases the amount available for synthesis of 5-hydroxytryptamine in the brain. 

The biosynthesis of tryptophan occurs by condensation of L-serine with indole, this reaction is catalyzed by tryptophan synthetase. The enzyme is a pyridoxal-phosphate containing enzyme which catalyzes nucleophilic p-substitution reactions of amino acids. The p-hydroxyl group of serine is substituted by indole by the action of the enzyme. The reaction is thought to proceed via a ketimine intermediate (27) which undergoes elimination to give an aminoacrylate-pyridoxal phosphate Schiff base (28). Addition at the P-carbon of indole followed by reversal of the process constitutes the enzymatic synthesis of L-tryptophan. 

Tryptophan synthase (EC 4.1.2.20) normally catalyzes the synthesis of tryptophan from serine by the oc,p elimination-addition reaction outlined in Scheme 5 where X = OH and Z = indole. The B protein of the oligomeric enzyme will catalyze the dehydration of serine, and in the presence of PLP and mercaptoethanol, the intermediate 15 will form adduct 25. This will then react as in Scheme 9 to yield the ketoacid 26 and pyridoxamine-phosphate 6. The net transamination has been shown to involve protonation at the 4 -Si face in yielding PMP (30). When the apoenzyme of tryptophan synthase is reconstituted with the unnatural substrates (4 / )- or (4 S)-[4- H,]pyridox-amine-phosphate and indole-3-pyruvic acid, an unnatural transamination... 

In the tissues, vitamin Be occurs predominantly as the phosphate of pyridoxal or pyridoxamine, especially the former, except in the liver. Pyri-doxal phosphate functions as a coenzyme in four types of reactions decarboxylation of amino acids, transamination, and the synthesis and cleavage of tryptophan (Chapter 19). This coenzyme is necessary for the deamination of amino acids and for the formation of urea nitrogen. It appears to be essential for the conversion of tryptophan to the pyridine coenzymes. Pyridoxine may be related to fatty acid metabolism and seems to be necessary for normal adrenal cortical function. ... 

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