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Figure 1 Overview

Tryptophan Synthase catalyzes the final two steps in the biosynthesis of the amino acid L-Tryptophan.  The enzyme is found in a variety of bacteria, fungi, and plants.   The enzyme exist as an a2b2 quartenary structure.  The Tryptophan Synthase displayed is the K87T mutant form, with only the ab dimer visible.

Tryptophan Synthase is an ideal model for illustrating the complex protein-protein interactions that occur between biomolecules.  Each subunit communicates with the other cooperatively to maximize the catalytic rate of the synthesis of L-tryptophan from indole-3-glycerol-phosphate.  The overall synthesis is completed by the substrate binding to the active site in the a-subunit, the product then channeled to the b-subunit active site, and then the product released.
 
 

Figure 2 a-subunit

The alpha subunit has an a/b barrel, which is formed from eight parallel beta strands with eight parallel a-helicies packed around it.  (

Figure 3)  The active site, which is composed of two main residues, ( Figure 4) Asp60 and Glu49, bind the substrate indole-3-glycerol-phosphate.  This substrate enters through an elongated random coil which has been shown to have very flexible conformations due to the individual residues forming this coil.  (The coil has been cleaved in the mutant form, but the start and stop residues of the coil are highlighted ( Figure 5)).  This coil moves approximately 7 Angstroms, from an open to a closed form, allowing the entrance or blocking of the substrate.

In the active site of the a-subunit, the substrate indole-3-glycerol-phoshate is converted to indole and 3-glyceraldehyde-phosphate.  The indole is then intramolecularly channeled to the b-subunit for further synthesis.  To view the mechanism of the a-subunit, click here.
 
 

Figure 6 b-subunit

The b-subunit consists of two domains called the N-terminal domain and C-terminal domain.  (

Figure 7)  The binding site for the coenzyme PLP (pyridoxal-5-phosphate) is sandwhiched between these two domains.   ( Figure 8). Once PLP is bound to the enzyme at Lys87, the enzyme is thermodynamically stabilized by "tightening" of the protein structure, essentially activating it.  The  PLP also participates in the mechanism of the conversion of indole to L-tryptophan.

The K87T mutant form seen here, has L-tryptophan bound to the active site. (

Figure 9).   This mutant has Threonine in place of Lysine at residue 87, which is the binding site of PLP.  This mutation causes the enzyme to irreversibly bind L-tryptophan, allowing the enzyme to be crystallized with L-tryptophan in the active site.

The Lys87 bound PLP forms a Schiff base with L-serine, preparing the L-serine for nucleophillic attack by indole.  The indole is activated by a basic reisdue pulling away the proton at N-1 of the indole.
 
 

Figure 10 ab Channel

 One of the most impressive features of tryptophan synthase is the intramolecular tunnel or channel from the a-subunit active site to b-subunit active site.  The two active sites are approximately 25 Angstroms apart, and the indole product from the a-subunit is channeled to the b-subunit active site.  This is essential, because if the indole was allowed to go free in solution, it could be diffused out of the cell, rendering the synthesis less effective.
 
 

Figure 11 ab Communications

The substrates and ligands that bind to the various sites of tryptophan synthase cause conformational changes not only in that particular subunit, but also in the other subunits, making the complete mechanism cooperative.  In one such scenario (as that illustrated by Figure 11), the PLP activated enzyme binds the substrate indole-3-glycerol-phosphate in the a-subunit active site.  Once IGP is bound, the a-subunit becomes closed, preventing it from allowing anymore IGP to enter.  At the same time, the b-subunit is activated to bind L-Serine to PLP in the form of the Schiff base.  The indole is then channeled from the a-subunit to the b-subunit active site.  This activates the a-subunit to the open form which allows the release of the glyceraldehyde-3-phophate side product.  Once the L-tryptophan is synthesized in the b-subunit, it is released, and the whole process starts over.

References

Subcellular Biochemistry, Volume 24: Proteins, Structure, Function and Engineering, pg. 207-244, edited by B.B. Biswas and Siddhartha Roy, Picnum Press, New York, 1994.

Miles, E., Rhee, S., and Davies, D. 1999. The Molecular Basis of Substrate Channeling, J. Biol. Chem., 274: 12193 - 12196.

Ahmed, S. A., Ruvinov, S. B., Kayastha, A. M., and Miles, E. W., 1991,  Mechanism of mutual activation of the tryptophan synthase a and b subunits.  Analysis of the reaction specificity and substrate-induced inactivation of active site and tunnel mutants of the b-subunit, J. Biol. Chem., 266: 21540-21557.