L-Tryptophan synthetase

Other lyases Pyridoxal phosphate L-tyrosine phenol lyase L-tryptophan synthetase... 

However, the a and p2 sub-units were shown to combine when mixed and in combination as the fully associated L-tryptophan synthetase a 2 complex they catalysed a reaction which is the sum of (0 and (h) but in which indole (40)—a product of (0 and a substrate for ( )—was not detectable as a free intermediate. Thus indole (40), although it can enter the pathway between indoleglycerol-3-phos-phate (38) and L-tryptophan (3) either as a growth supplement or an accumulated product, may not be a true physiological intermediate. Yanofsky and his collaborators have observed that the full a/ 2 complex has an enhanced catalytic activity relative to the component a and P2 sub-units and that there is a concomitant gain in specificity for the reaction (ii). Thus the a 2a complex was 100 times more active than the a sub-unit in reaction (i) and 30 times more active than the 2 sub-unit in reaction (ii). In addition these workers observed that the intermediate 0. 2 complex was about one half as active per p2 sub-unit as the tetrameric complex and they concluded that each a unit contributed equally to each of the two identical active sites in the full a 2 enzyme. During the process of synthesis of the complex it has been assumed that the /S chains dimerise rapidly and then become associated with the a chains to give the tetrameric species. 

In addition to the striking effects of sub-unit interaction upon catalytic activity and specificity which have been noted for the L-tryptophan synthetase from Escherichia coli the enzyme and its a and 2 sub-units have been shown to catalyse a variety of other reactions not entirely connected with L-tryptophan biosynthe-... 

The L-tryptophan synthetases of fungal origin which have been studied appear to be quite different from the bacterial enzyme. A single undissociable protein species catalyses the biosynthetic reaction from indoleglycerol phosphate (38) to L-tryptophan (3) and the reactions (z) and and a mutant of Newospora... 

Enzyme S was quite specific for DMAPP and cAATrp, neither IPP, GPP, Trp nor A-acetyltryptophan being used as substrates. In this connection it is of interest to note that another secondary transferase, 4-dimethylallyl-L-tryptophan synthetase, utilizes Trp as a cosubstrate with DMPP en route to the clavine alkaloids (Heinstein et al., 1971 Lee, 1974), while in this case Trp did not even bind to the enzyme. The specificity of S for a cyclized Trp may be compared with that established by Allen (1972) for cyclo-L-alanyl-2-(l, l-dimethylallyl)-L-tryptophanyl synthetase. In this case, however, the substrate, cyclo-L-alanyl-L-tryptophanyl, could be replaced by cyclo-L-prolyl-L-tryptophanyl (Deyrup and Allen, 1975). 

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... 

In the presence of both the substrates, indole-3-glycerolphosphate and L-serine, tryptophan synthetase, 02)82 catalyses two parallel reactions. The a subunits in the complex carry out the cleavage of indole-3-glycerolphosphate releasing glyceralde-hyde-3-phosphate in the medium but the indole remains bound to the complex. Concomitantly, the /Sj subunits promote the conversion of serine into the aminoacrylate-pyridoxal-P species (Fig. 27, 1) which acts as a Michael acceptor for the indole producing the L-tryptophan-pyridoxal-P complex from which the amino acid is released by hydrolysis (Fig. 27). That the aforementioned molecular events can be described in such vivid detail is due to an ingenious series of partial reactions... 

Although the tryptophan synthetase and tryptophanase reactions have been the best studied replacement and 0 eUmination-deamination reactions, others pf special interest are D-serine dehydratase [75-77] from E. coli, D-threonine dehydratase and L-threonine dehydratase from Serratia marcescens [78]. The only information available on the above enzymes is that in these cases also, the events at occur with retention of configuration. 

It is a monomeric protein of M.W. about 70,000, shows Kj, values for L-tryptophan and dimethylallyl pyrophosphate of 0.067 and 0.2 mM, respectively, and seems to have a relatively low turnover number, about 7 sec . During studies on this enzyme it was observed (13) that agroclavlne and elymoclavine, the terminal alkaloids in the strain used for the isolation of the enzyme, inhibited purified DMAT synthetase. At concentrations of 3 mM ( v<750 mg/1) agroclavlne and elymoclavine inhibited the enzyme 90% and 70%, respectively. The inhibition is of a mixed or uncompetitive type as shown by kinetic analysis with either tryptophan or dimethylallyl pyrophosphate as the variable substrate (Fig. 6). Subsequently, feedback Inhibition by elymoclavine was also demonstrated by GrSger s group (3) for chanoclavlne cyclase and by us for anthranllate synthetase from... 

The biosynthesis of ergot alkaloids is initiated by prenyla-tion of tryptophan with dimethylallyl pyrophosphate (DMAPP) to yield 4-dimethylallyltryptophane (DMAT) (4) (Fig. 35.2) (Floss, 1980). This alkylation step is believed to be rate limiting, and the enzyme that catalyzes this reaction, DMAT synthetase, has been purified more than 60 times (Maier and Groger, 1976 Waller and Dermer, 1981). The enzyme appears to be specific for L-tryptophan and is inhibited by agroclavine (2) and elymoclavine (5) the end products of the reaction. 

Fig. 1. The tryptophan biosynthetic pathway. Abbreviations AS, anthranilate synthetase PRPP, 5-phosphoribosyI-l-pyrophosphate PRT, phosphoribosyi transferase PRAI, phosphoribosyi anthranilate isomerase InGPS, indoleglycerol phosphate synthetase TS, tryptophan synthetase TS-a, tryptophan synthetase a-chain subunit TS- z, tryptophan synthetase -chain dimer subunit.

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]. 

The stereochemistry of the a,j0-desaturation of cryptoechinulin was reported by Fuganti et al. and is summarized in Scheme 35 [59]. These workers prepared L-tryptophan stereospecifically labeled with tritium in the -methylene position from labeled serine, indole, and fibre-entrapped tryptophan synthetase obtained from E. coli. When (3 i )[3 - H 3 - C]-L-tryptophan was fed to Aspergillus amstelodami, incorporation into echinulin and cryptoechinulin took place with 95% and 98% retention of tritium activity, respectively. Feeding of (3 S)[3 - H 3 - C]-L-tryptophan to Aspergillus amstelodami gave incorporation into echinulin and cryptoechinulin with 96% and 5% retention of tritium activity, respectively. Thus, in the desaturation reaction the pro-S hydrogen is stereospecifically removed. 

Tryptophanyl-tRNA synthetase l-Tryptophan tRNA ligase (AMP-forming) 6.1.1.2 237... 

During this period I was frustrated by my inability to obtain tryptophan synthetase in pure form from Neurospora. Therefore, I searched for a more suitable enzyme for studies on protein structure alterations resulting from mutations. I characterized d- and L-serine deaminases from Neurospora - with this objective in mind but was unable to develop a convenient isolation procedure for mutants lacking these enzymes and so stopped these investigations. 

During my stay at Western Reserve University I also continued my studies of tryptophan synthetase—but now with the enzyme from E. coU as well as the one from Neurospora. With Jody Stadler and Martin Rachmeler initial observations were made that suggested that the tryptophan synthetase reaction was more complex than we had imagined. The enzyme converted indole-3-glycerol phosphate to indole and indole -3-glycerol phosphate to tryptophan in addition to catalyzing the previously characterized reaction, indole -F l-serine —> L-tryptophan< - - >. 

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