Tryptophan reaction with pyridoxal

The second part of the reaction requires pyridoxal phosphate (Fig. 22-18). Indole formed in the first part is not released by the enzyme, but instead moves through a channel from the a-subunit active site to the jS-subunit active site, where it condenses with a Schiff base intermediate derived from serine and PLP. Intermediate channeling of this type may be a feature of the entire pathway from chorismate to tryptophan. Enzyme active sites catalyzing different steps (sometimes not sequential steps) of the pathway to tryptophan are found on single polypeptides in some species of fungi and bacte-... 

Fig. 7.1 Reactions catalyzed at the active sites of the a subunit (a reaction) and of the 0 subunit (0 reaction) and the coupled, physiological reaction (a0 reaction). In the a0 reaction, indole produced by cleavage of indole-3-glycerol phosphate at the a site diffuses through an intramolecular tunnel to the 0 site 25-30 A distant where it undergoes a pyridoxal phosphate-dependent /3-replacement reaction with L-serine to form L-tryptophan. Abbreviations used IGP, indole-3-glyceroI phosphate G-3-P, o-glyceraldehyde 3-phosphate, IND, indole [IND], indole intermediate PLP, pyridoxal phosphate.

At first glance, Eq. (15) appears too complex to allow measurement of individual reaction rate constants. However, as we illustrate with this example, it is possible to extract estimates of all four rate constants from an analysis of the concentration dependence of the observed rates. The time dependence of reaction of serine with pyridoxal phosphate at the /3-site of tryptophan synthase provides a good example of two-step reaction kinetics because of the unique optical... 

Finally, the three-carbon side chain of the indole-3-glycerolphosphate is removed to produce indole itself and glyceraldehyde-3-phosphate (Scheme 12.25). The indole moves from one subunit of the enzyme, tryptophan synthase (EC 4.2.1.20), to another where it encounters the result of dehydration of a pyridoxal-bound serine (Ser, S) to which it adds. Then, hydrolytic cleavage of the resulting pyridoxal derivative of tryptophan (Trp, W) yields pyridoxal, ready for another reaction with, for example, serine (Ser, S) and tryptophan (Trp, W). 

The pyridoxal/pyridoxamine system is not only involved in transaminations. We examined catalysts for the synthesis of tryptophan by coupling reactions with indole and for pyridoxal-catalyzed aldol condensations.We also examined optical induction in... 

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 ... 

Various bacteria, E. coli in particular, cleave the side chain of tryptophan to yield indole S76). This putrefactive reaction is the source of indole and skatol in man. In earlier times these compounds were of great interest because they were believed to represent a serious health problem. Studies of this reaction with the enzyme tryptophanase has shown that indole, ammonia and pyruvic acid are the reaction products (377) and that the coenzyme for the reaction is pyridoxal phosphate... 

Biosynthesis of some classes of terpene indole alkaloids is well understood. In certain cases, many of the enzymes that are responsible for biosynthesis have been cloned and mechanistically studied. In other cases, biosynthesis pathway is only proposed based on the results of feeding studies with isotopically labeled substtates and from the structures of isolated biosynthetic intermediates. All terpene indole alkaloids are derived from tryptophan and the iridoid terpene secologanin (Fig. 14.11). Tryptophan decarboxylase, a pyridoxal-dependent enzyme [29], converts tryptophan to tryptamine [30]. The following strictosidine synthase-catalyzed Mannich reaction connects ttyptamine and secologanin to yield strictosidine [31]. The Apocynaceae, Loganiaceae, Rubiaceae, and Nyssaceae families of plants each produce terpene indole alkaloids with dramatically diverse structures [32-34]. The mechanisms and control of... 

Applications of tryptophan synthetase Tryptophan synthetase (EC 4.2.1.20) is a pyridoxal phosphate-dependent enzyme that, in the cell, catalyzes the a,/3-elimination of water from serine to form a pyridoxyl-bound a-aminoacrylate, which undergoes Michael addition of indole to form the named amino acid. This type of reaction has been used to prepare (5)-tryptophan isotopomers with a variety of labeling patterns by use of different labeled indoles and (5)-serines in yields of up to 98% based on indole and 92% based on (5)-serine. 

Numerous examples of modiflcations to the fundamental cyclodextrin structure have appeared in the literature.The aim of much of this work has been to improve the catalytic properties of the cyclodextrins, and thus to develop so-called artificial enzymes. Cyclodextrins themselves have long been known to be capable of catalyzing such reactions as ester hydrolysis by interaction of the guest with the secondary hydroxyl groups around the rim of the cyclodextrin cavity. The replacement, by synthetic methods, of the hydroxyl groups with other functional groups has been shown, however, to improve remarkably the number of reactions capable of catalysis by the cyclodextrins. For example, Breslow and CO workersreported the attachment of the pyridoxamine-pyridoxal coenzyme group to beta cyclodextrin, and thus found a two hundred-fold acceleration of the conversion of indolepyruvic acid into tryptophan. 

This enzyme [EC 2.6.1.1] (also known as transaminase A, glutamicioxaloacetic transaminase, and glutamic aspartic transaminase) catalyzes the reversible reaction of aspartate with a-ketoglutarate to produce oxaloace-tate and glutamate. Pyridoxal phosphate is a required cofactor. The enzyme has a relatively broad specificity, and tyrosine, phenylalanine, and tryptophan can all serve as substrates. 

This pyridoxal-phosphate-dependent enzyme [EC 4.2.1.20] catalyzes the reaction of L-serine with l-(indol-3-yl)glycerol 3-phosphate to produce L-tryptophan and glyceraldehyde 3-phosphate. The enzyme will also catalyze (a) the conversion of serine and indole into tryptophan and water and (b) conversion of indoleglycerol phosphate into indole and glyceraldehyde phosphate. 

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). 

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

Tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) belongs to the family of the pyridoxal 5 -phosphate (PLP)-dependent enzymes. It serves in vivo to degrade L-tryptophan, is induced by L-tryptophan, and found in various bacteria, particularly in enteric species. Tryptophanase catalyzes a,/3-elimination1 and /3-replacement reactions on interaction with L-tryptophan and various other /3-substituted amino acids2 ... 

We next synthesized the pyridoxal-bound fl-CD catalyst 44 (Scheme 2.8) [43], which produced 3-5 times more tryptophan when incubated with indole, [f-chloroalanine, and A12(S04)3 (pH 5.2 and 100°C) than the reaction in which the pyridoxal derivative was replaced by simple pyridoxal. However, tryptophan yield was still only a few percent. As expected, this kinetic advantage disappeared at higher indole concentrations due to saturation of the binding site. Furthermore, L-tryptophan was produced in ca. 10% excess relative to the D-enantiomer. 

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-... 

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... 

A series of a-aminoesters has been hydrolyzed to a-amino acids using alcalase in the presence of pyridoxal 5-phosphate[40). During the course of these reactions, the amino acids precipitated from the reaction mixture, thereby protecting them from racemisation. The method was used to prepare enantiomerically enriched phenylalanine, leucine, tryptophan and norvaline with high selectivity (Fig. 9-19). 

Combined use of microbial enzymes as biocatalysts with chemical synthesis has its origin in the steroid transformation developed in the USA in the early 1950s. Arima and his group [11] invented a unique microbial conversion process, in which the aliphatic side-chain of cholesterol was cleaved to produce a steroid core as a starting material for chemical synthesis of steroid hormones. Yamada et al. discovered the reverse reaction of the pyridoxal-containing L-amino acid lyases and applied them to synthesize L-tryptophan and l-DOPA [12] from pyruvate, ammonia and corresponding aromatic compounds. Since these early achievements, a variety of unique processes with newly screened microbial enzymes as biocatalysts have been invented. 

An unusual variant of a /8 elimination reaction is shown by kyneurinase which catalyses the production of L-alanine and 3-hydroxyanthranilate from 3-hydroxy kyneurine (Fig. 33), an intermediate in tryptophan biosynthesis. Although the reaction has not been subjected to mechanistic investigation a pathway consistent with the previously demonstrated properties of pyridoxal-P to stabilise a j8-carban-ion is illustrated in Fig. 34. 

Studies with tryptophan synthetase [95] and tryptophanase [96] highlight the strength as well as the weakness of the spectroscopic approach as a tool for the identification and kinetic analysis of intermediary species produced in pyridoxal-P-dependent reactions. The involvement of the species of types 1, 2 and 4 (Fig. 31) in tryptophanase catalysed reactions and the kinetics of their formation have been extensively studied spectroscopically [96], but the quinonoid intermediate (2) and the acrylyl pyridinium species (4) could not be discerned from each other because the two chromophores absorbed in the same region at about 500 nm. 

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... 

Indoleglycerol 3-phosphate (13) is converted into tryptophan (14) by the action of L-tryptophan synthase. The mechanism of this enzymatic reaction involves formation of a Schiff base with an enzyme-bound pyridoxal phosphate. The a-aminoacrylate Schiff base formed undergoes the addition of a p-substituent to produce tryptophan (Floss, 1986) (Fig. 7.5). 

In the presence of the reconstituted 0202 complex, the rates of these partial reactions are 30 to 100 times greater than with the individual subunits. The sum of the two partial reactions Indole 3-glycerol phosphate + L-serine - L-tryptophan + 3-phosphoglyceralde-hyde + H2O, is catalysed by the O202 complex, and free indole is not detectable as an intermediate in this overall reaction. T.s. from E. coli and other prokaryotes therefore serves as a simple and very effective model of a multienzyme complex. Pyridoxal phosphate is essential for enzymatic activity each 0 subunit binds one molecule of the coenzyme. Hie reactions proceed via the SchifTs base between the enzyme-bound pyridoxal phosphate and amino acrylate (structure c in the scheme shown in the entry 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... 

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