Indole 3-glycerol phosphate tryptophan intermediate

The two intermediates in the conversion of anthranilate to indole-3-glycerol phosphate, phosphoribosylan-thranilate and l -(O-carboxyphenylamino)-1 -deoxyribu-lose-5 phosphate, were originally postulated to account for the involvement of phosphoribosyl pyrophosphate in indole-3-glycerol phosphate formation. Support for the postulate was obtained when the dephosphorylated derivative of the second of these intermediates was found in the culture fluids of certain tryptophan-requiring bacterial mutants. The corresponding derivative of the first intermediate has not been found, probably because of its instability. Indeed, this compound, when formed in extracts, is rapidly broken down to anthranilate and ribose-5-phosphate. 

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... 

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.

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... 

The a subunit catalyzes the formation of indole from indole-3-glycerol phosphate, whereas each P subunit has a PLP-containing active site that catalyzes the condensation of indole and serine to form tryptophan. The overall three-dimensional structure of this enzyme is distinct from that of aspartate aminotransferase and the other PLP enzymes already discussed. Serine forms a Schiff base with this PLP, which is then dehydrated to give the Schiffbase of aminoacrylate. This reactive intermediate is attacked by indole to give tryptophan. 

In B. subtilis, the pathway from chorismate to tryptophan is feedback-inhibited by tryptophan, which suppresses anthranilate synthase activity. Mutant B. subtilis that lacks tryptophan synthetase can grow on minimal medium only when supplemented with exogenous tryptophan. Under these conditions, none of the intermediates in the tryptophan biosynthetic pathway from anthranilate to indole 3-glycerol phosphate are produced. However, when the bacteria have depleted the medium of tryptophan, the levels of those intermediates increase, even though there is no net production of tryptophan. Why ... 

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... 

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