Substrate channeling is a process by which two sequential enzymes interact to transfer a metabolite (or intermediate) directly from one enzyme active site to the next without allowing free diffusion of the metabolite. There are numerous examples of sequential enzyme pairs which are thought to exhibit channeling in glycolysis and in several biosynthetic pathways and channeling is thought to play an important role in metabolic regulation and cellular modulation of enzymatic activities. Tryptophan synthase, the final enzyme is tryptophan biosynthesis, is considered the best example for substrate channeling. The enzyme exists as an alpha2Beta tetramer with alpha and Beta subunits each catalyzing a distinct chemical reaction such that the physiological alpha Beta reaction leads to the overall conversion of indole 3-glycerol phosphate (IGP) and serine to tryptophan, glyceraldehyde-3-phosphate, and H20. In the absence of serine, IGP is cleaved to indole and glyceraldehyde-3phosphate at the alpha site, although at a slow rate. The Beta subunit catalyzes the pyridoxal-dependent reaction of indole and serine to form tryptophan. These data suggest that indole is an intermediate in the alpha/Beta reaction although indole has never been observed as an intermediate in the physiological alpha/Beta reaction. In fact, the failure to detect or trap indole during net conversion of IGP to tryptophan has led to the suggestion that indole is channeled from the alpha to the Beta subunit. Further support for channeling is provided by the recently solved three-dimensional x-ray structure of tryptophan synthase which revealed the presence of a 25 Angstroms long hydrophobic channel or connecting the alpha and Beta subunits. Although there is chemical and structural evidence suggestive of indole channeling, until now the kinetics of the reaction have not been investigated in sufficient detail to establish whether channeling occurs and to determine what elementary steps of the reaction pathway are responsible for channeling. Studies from the PI's lab have recently provided the first definitive kinetic evidence for the channeling of indole and have lead to the discovery of a novel triggering mechanism by which the alpha and Beta subunits communicate over the 25 Angstroms distance between their active sites. The goals of the current proposal are: (1) Explore the structural basis for substrate channeling and alpha/Beta intersubunit communication by time-resolved x-ray crystallography and solid-state NMR spectroscopy (2) Relate the structure of the enzyme to the dynamics of catalysis and intersubunit communications by a complete kinetic and thermodynamic analysis of enzymes containing mutations in key residues.

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National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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Yale University
Schools of Medicine
New Haven
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