STRUCTURAL BASIS OF THE ALLOSTERIC INTERACTIONS IN TRYPTOPHAN SYNTHASE REVEALED BY KINETIC CRYSTALLOGRAPHY

Michael Weyand¹, Thomas R. Schneider², Ilme Schlichting¹

¹ Max-Planck-Institute for Molecular Physiology, Dep. of Physical Biochemistry, Rheinlanddamm 201, D-44139 Dortmund, Germany;
² Goettingen University, Institute for Inorganic Chemistry, Tammannstr. 4, D-37077 Goettingen, Germany

Tryptophan Synthase (TRPS) is an alpha2beta2 tetrameric enzyme complex which catalyses the last two steps in the synthesis of L-tryptophan. This physiological important alpha-beta-reaction involves the conversion of indole 3-glycerol phosphate (IGP) and serine to tryptophan and water. During the enzymatic reaction an aminoacrylate intermediate (A-A) is formed at the beta- site, which activates the alpha-reaction. TRPS is considered a classic example of an enzyme that exhibits substrate channeling, where a metabolic intermediate is transferfed from one active site to another without free diffusion.

Direct and global information at the atomic level about the mechanism and structural dynamics for such allosteric signaling can be obtained from two different crystallogrpahic methods: Structures can be determined by time- resolved crystallography on fast time scales using the Laue method or by kinetic crystallography. For the investigations of the allosteric interactions of TRPS, we chose the latter for reasons that will be discussed. A flow cell was used to generate the aminoacrylate intermediate of the beta- reaction in the TRPS crystals under steady state conditions in the presence of serine and the alpha-site inhibitors indole propanol phosphate (IPP) or 5-fluorindole propanol phosphate (F-IPP). Since the aminoacrylate is generated much faster than its decays, it accumulates under steady state conditions and is therefore amenable to kinetic crystallographic approaches.

Following this techniques we have recently determined and compared the structures of F-IPP complexed TRPS with and without the aminoacrylate intermediate. On the basis of these structures, a pathway for communication between the alpha- and beta-active sites has been established. Proving these results structure investigations of TRPS mutants using the same kinetic crystallographic approach are in progress.