4- tryptophan synthase

Edith Wilson Miles, Syed Ashrafuddin Ahmed, C. Craig Hyde, Arvind M. Kayastha, Xiang-Jiao Yang, Sergei B. Ruvinov, and 

National Institute of Diabetes and Digestive and Kidney Diseases 

National Institutes of Health Building 8, Room 2A09, Bethesda, MD 20892 

Tryptophan synthase (EC 4.2.1.20) from bacteria is a classic multienzyme complex that channels a metabolic intermediate (for reviews and commentaries see 1-6)). Structure/ function analysis of tryptophan synthase was pioneered by genetic and biochemical investigations beginning in the mid-1940 s. This chapter emphasizes the relationship between the function of tryptophan synthase and the three-dimensional structure of the tryptophan synthase 02)82 complex from Salmonella typhimurium7,8) and focuses on studies carried out since a recent review.6  

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 

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Hyde, C.C., et al. Three-dimensional structure of the tryptophan synthase az pz multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 263 17857-17871, 1988. 

Oligomeric enzymes may also carry out different but related reactions on different subunits. Thus, tryptophan synthase is a tetramer consisting of pairs of different subunits, Purified a-subunits catalyze the following reaction ... 

Figure 1. Schematic outline of various products and associated enzymes from the shikimate and phenolic pathways in plants (and some microorganisms). Enzymes (1) 3-deoxy-2-oxo-D-arabino-heptulosate-7-phosphate synthase (2) 5-dehydroquinate synthase (3) shikimate dehydrogenase (4) shikimate kinase (5) 5-enol-pyruvylshikimate-3-phosphate synthase (6) chorismate synthase (7) chorismate mutase (8) prephenate dehydrogenase (9) tyrosine aminotransferase (10) prephenate dehydratase (11) phenylalanine aminotransferase (12) anthranilate synthase (13) tryptophan synthase (14) phenylalanine ammonia-lyase (15) tyrosine ammonia-lyase and (16) polyphenol oxidase. (From ACS Symposium Series No. 181, 1982) (62).

Substrate and inhibitor of tryptophan synthase and 78 other enzymes... 

This enzyme [EC 2.4.2.18], also referred to as phospho-ribosyl-anthranilate pyrophosphorylase, catalyzes the reaction of anthranilate with phosphoribosylpyrophos-phate to produce A-5 -phosphoribosylanthranilate and pyrophosphate. In certain species, this enzyme is part of a multifunctional protein, together with one or more other components of the system for the biosynthesis of tryptophan (i.e., indole-3-glycerol-phosphate synthase, anthranilate synthase, tryptophan synthase, and phos-phoribosylanthranilate isomerase). 

ACYL-SERINE INTERMEDIATE CHYMOTRYPSIN CATALYTIC TRIAD TRYPTOPHANASE TRYPTOPHAN SYNTHASE T state,... 

Yutani, K., Ogasahara, K., Tsujita, T. and Sugino, Y. (1987) Dependence of conformations stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase subunit. Proc. Natl. Acad. Sci. USA, 84, 4441-4444. 

In the tryptophan branch (Fig. 22-17), chorismate is converted to anthranilate in a reaction in which glutamine donates the nitrogen that will become part of the indole ring. Anthranilate then condenses with PRPP. The indole ring of tryptophan is derived from the ring carbons and amino group of anthranilate plus two carbons derived from PRPP. The final reaction in the sequence is catalyzed by tryptophan synthase. This enzyme has an a2/32 subunit structure and can be dissociated into two a subunits and a /32 subunit that catalyze different parts of the overall reaction ... 

MECHANISM FIGURE 22-18 Tryptophan synthase reaction. This enzyme catalyzes a multistep reaction with several types of chemical rearrangements. An aldol cleavage produces indole and glyceraldehyde 3-phosphate this reaction does not require PLP. Dehydration of serine forms a PLP-aminoacrylate intermediate. In steps and this condenses with indole, and the product is hydrolyzed to release tryptophan. These PLP-facilitated transformations occur at the /3 carbon (C-3) of the amino acid, as opposed to the a-carbon reactions described in Figure 18-6. The /3 carbon of serine is attached to the indole ring system. Tryptophan Synthase Mechanism... 

Pan P., Woehl, E., Dunn, M.F. (1997) Protein architecture, dynamics and allostery in tryptophan synthase channeling. Trends Biochem, Sci, 22, 22-27. 

Effect of mRNA and Protein Stability on Regulation E. coli cells are growing in a medium with glucose as the sole carbon source. Tryptophan is suddenly added. The cells continue to grow, and divide every 30 min. Describe (qualitatively) how the amount of tryptophan synthase activity in the cells changes with time under the following conditions ... 

Beta replacement is catalyzed by such enzymes of amino acid biosynthesis as tryptophan synthase (Chapter 25),184 O-acetylserine sulfhydrylase (cysteine synthase),185 186a and cystathionine (3-synthase (Chapter 24).187 188c In both elimination and (3 replacement an unsaturated Schiff base, usually of aminoacrylate or aminocrotonate, is a probable intermediate (Eq. 14-29). Conversion to the final products is usually assumed to be via hydrolysis to free aminoacrylate, tautomerization to an imino acid, and hydrolysis of the latter, e.g., to pyruvate and ammonium ion (Eq. 14-29). However, the observed stereospecific addition of a... 

A (3 replacement reaction catalyzed by the PLP-dependent tryptophan synthase converts indoleglycerol phosphate and serine to tryptophan. Tryptophan synthase from E. coli consists of two subunits associated as an a2P2 tetramer (Fig. 25-3). The a subunit catalyzes the cleavage (essentially a reverse aldol) of indoleglycerol phosphate to glyceraldehyde 3-phosphate and free indole (Fig. 25-2, step s).67 The P subunit contains PLP. It presumably generates, from serine, the Schiff base of aminoacrylate, as indicated in Fig. 25-2 (step f). The enzyme catalyzes the addition of the free indole to the Schiff base to form tryptophan. The indole must diffuse for a distance of 2.5 ran... 

Figure 25-3 The structure of the two-enzyme a2p2 complex tryptophan synthase.65 66 The view is with the twofold axis of the OC2P2 complex vertical with the two a subunits at the ends and the P subunits in the center. The tunnel through which indole molecules released from indole propanol phosphate (IPP) in the a subunits to the pyridoxal phosphate (PLP) in the p subunits is shaded. Courtesy of C. Craig Hyde and Edith Wilson Miles.

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