Received November 27, 2000

Revised Manuscript Received January 23, 2001

Abstract:

The tryptophan synthase bienzyme complex is the most extensively documented example of substrate channeling in which the oligomeric unit has been described at near atomic resolution. Transfer of the common metabolite, indole, between the - and the -sites occurs by diffusion along a 25-Å-long interconnecting tunnel within each -dimeric unit of the ₂₂ oligomer. The control of metabolite transfer involves allosteric interactions that trigger the switching of -dimeric units between open and closed conformations and between catalytic states of low and high activity. This allosteric signaling is triggered by covalent transformations at the -site and ligand binding to the -site. The signals are transmitted between sites via a scaffolding of structural elements that includes a monovalent cation (MVC) binding site and salt bridging interactions of Lys 167 with Asp 305 or Asp 56. Through the combined strategies of site-directed mutations of these amino acid residues and cation substitutions at the MVC site, this work examines the interrelationship of the MVC site and the alternative salt bridges formed between Lys 167 with Asp 305 or Asp 56 to the regulation of channeling. These experiments show that both the binding of a MVC and the formation of the Lys 167-Asp 56 salt bridge are important to the transmission of allosteric signals between the sites, whereas, the salt bridge between K167 and D305 appears to be only of minor significance to catalysis and allosteric regulation. The mechanistic implications of these findings both for substrate channeling and for catalysis are discussed.

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