Serine tryptophan synthesis

The fifth and terminal step of tryptophan synthesis, the removal of the glycerolphosphate side chain and its replacement by the alanyl moiety of L-serine [Fig. 4 (17)], is catalyzed by the structurally complex enzyme tryptophan synthase (TS). This protein, probably the earliest known multicomponent enzyme, has been extensively studied and often reviewed (Crawford, 1975). The complex nature of the enzyme is illustrated by its capability of catalyzing the following distinct reactions ... 

Serine, tryptophan and histidine donate 1-carbon units to folate metabolites that are used for DNA and RNA synthesis during cell division and growth. NB Vitamin B12 is needed for the metabolism and function of folate. 

L-Tryptophan synthase, tryptophan desmolase, L-serine hydro-lyase (adding imhleglycerol-phos-phate) (EC 4.2.1.20) the enzyme catalysing the synthesis of L-tryptophan from L-serine and indole 3-glycerol phosphate. T.s. from E. coli (M, 149,000) and other prokaryotes has 02 subunit composition. The enzyme separates easily into monomeric subunit a (also called protein B, M, 29,000) and dimeric subunit 02 (also called protein B Af, of dimer 90,000) when eluted from DEAE cellulose with a sodium chloride gradient. The separated subunits catalyse partial reactions of L-tryptophan synthesis ... 

The fifth enzyme activity, tryptophan synthetase [EC 4.2.1.20, L-serine hydro-lyase (adding indole)], catalyzes the final step in tryptophan synthesis. It consists of a complex of two nonidentical protein subunits, B (/ -chains) and A (a-chains) [7,14,38-41], which are coded for by the fourth and fifth tryptophan genes, respectively, in E. coli [14] and S. typhimurium [27]. The A subunit has been purified and shown to be a single polypeptide a-chain [42]. Wilson and Crawford [39] purified the B-protein subunit. Their conclusion that it was a dimer P2) which complexed with two a-chain subunits to form the tryptophan synthetase ( 2 Pi) is supported by other studies of the subunits and their association [40,41], The two proteins of tryptophan synthetase (TS) can catalyze the following reactions [38,43]. 

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). 

Since tryptophan synthase does not (or poorly) tolerate the substitution of indoles by large groups (iodo- and nitro-groups), such cases have lead to the development of a facial chemical synthesis of racemic A-oc-acetyltryptophan derivatives from 5- and 6-monosubstituted indoles and L-serine, followed by an enzymatic resolution step using acylase Amano resulting in substituted L-tryptophans in high yields and enantiomeric purity of 91-100% ee [66] (Fig. 4). 

Fig. 3 Enzymatic synthesis of L-tryptophans from indoies, L-serine and lysates of recombinant E. coli overexpressing tryptophan synthase (X = F, Cl, Br at carbon 4,5,6,7)...

The three-dimensional structure of the tryptophan synthase 02)82 complex from S. typhimurium reveals that the four polypeptide subunits are arranged in an extended a/8/Jo order forming a complex 150 A long.7 A schematic view of a single a/ ft pair based on the crystal structure is shown in the color plate . The 02)82 complex catalyzes the synthesis of L-tryptophan from indole-3-glycerol phosphate and L-serine, termed the a)3 reaction (Fig. 7.1). The a and )3 subunits can be separated and shown to catalyze two distinct reactions, termed the a and / reactions, respectively (Fig. 7.1). The rates of the a and / reactions are greatly increased when catalyzed by the 02)82 complex. Although the o)3 reaction is formally the sum of the a and )3 reactions, indole does not appear as a free intermediate in solution in this reaction.17-21 This result indicates that indole is a... 

Schiff base, or quinoidal intermediates do not appreciably affect the rate of the a reaction.90113 (2) L-serine and amino acids such as O-methyl-L-serine that form ES III do stimulate the a reaction.113 (3) The rate of indole-3-glycerol phosphate turnover is roughly correlated with the rate of formation of ES III for each of the amino acids.90-113 (4) The kinetics of the lag in cleavage of indole-3-glycerol phosphate and synthesis of L-tryptophan under single turnover conditions correspond to the rate of ES III formation... 

PAH, a nonheme iron-containing enzyme, is a member of a larger BI Independent amino acid hydroxylase family. In addition to PAH, the enzyme family includes tyrosine hydroxylase and tryptophan hydroxylase. The enzymes in this family participate in critical metabolic steps and are tissue specific. PAH catabolizes excess dietary PA and synthesizes tyrosine. In adrenal and nervous tissue, tyrosine hydroxylase catalyzes the initial steps in the synthesis of dihydrox-yphenylalanine. In the brain, tryptophan is converted to 5-hydroxytryptophan as the first step of serotonin synthesis. Consequently, these enzymes are highly regulated not only by their expression in different tissues but also by reversible phosphorylation of a critical serine residue found in regulatory domains of the three enzymes. Since all three enzymes are phosphorylated and dephosphorylated by different kinases and phosphatases in response to the need for the different synthetic products, it is not unexpected that the exact regulatory signal for each member of the enzyme family is unique. 

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... 

Purines and pyrimidines are derived largely from amino acids. The biosynthesis of these precursors of DNA, RNA, and numerous coenzymes will be discussed in detail in Chapter 25. The reactive terminus of sphingosine, an intermediate in the synthesis of sphingolipids, comes from serine. Histamine, a potent vasodilator, is derived from histidine by decarboxylation. Tyrosine is a precursor of the hormones thyroxine (tetraiodothyronine) and epinephrine and of melanin, a complex polymeric pigment. The neurotransmitter serotonin (5-hydroxytryptamine) and the nicotinamide ring of NAD + are synthesized from tryptophan. Let us now consider in more detail three particularly important biochemicals derived from amino acids. 

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. 

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... 

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