Long-range interactions in the alpha subunit of tryptophan synthase help to coordinate ligand binding, catalysis, and substrate channeling

Jennifer M. Axe, David D. Boehr

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24 Citations (Scopus)

Abstract

The α-subunit of tryptophan synthase (αTS) catalyzes the conversion of indole-3-glycerol phosphate to d-glyceraldehyde-3-phosphate and indole. We propose that allosteric networks intrinsic to αTS are modulated by the binding of the β-subunit to regulate αTS function. Understanding these long-range amino acid networks in αTS thus gives insight into the coordination of the two active sites within TS. In this study, we have used Ala residues as probes for structural and dynamic changes of αTS throughout its catalytic cycle, in the absence of the β-subunit. Projection analysis of the chemical shift changes by site-specific amino acid substitutions and ligand titrations indicates that αTS has three important conformational states: ligand-free, glyceraldehyde-3-phosphate-bound(like), and the active states. The amino acid networks within these conformations are different, as suggested by chemical shift correlation analysis. In particular, there are long-range connections, only in the active state, between Ala47, which reports on structural and dynamic changes associated with the general acid/base Glu49, and residues within the β2α2 loop, which contains the catalytically important Asp60 residue. These long-range interactions are likely important for coordinating chemical catalysis. In the free state, but not in the active state, there are connections between the β2α2 and β6α6 loops that likely help to coordinate substrate binding. Changes in the allosteric networks are also accompanied by protein dynamic changes. During catalytic turnover, the protein becomes more rigid on the millisecond timescale and the active-site dynamics are driven to a faster nanosecond timescale.

Original languageEnglish (US)
Pages (from-to)1527-1545
Number of pages19
JournalJournal of Molecular Biology
Volume425
Issue number9
DOIs
StatePublished - May 13 2013

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Tryptophan Synthase
Catalysis
Ligands
Glyceraldehyde 3-Phosphate
Catalytic Domain
Amino Acids
Amino Acid Substitution
Proteins
Acids

All Science Journal Classification (ASJC) codes

  • Molecular Biology

Cite this

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title = "Long-range interactions in the alpha subunit of tryptophan synthase help to coordinate ligand binding, catalysis, and substrate channeling",
abstract = "The α-subunit of tryptophan synthase (αTS) catalyzes the conversion of indole-3-glycerol phosphate to d-glyceraldehyde-3-phosphate and indole. We propose that allosteric networks intrinsic to αTS are modulated by the binding of the β-subunit to regulate αTS function. Understanding these long-range amino acid networks in αTS thus gives insight into the coordination of the two active sites within TS. In this study, we have used Ala residues as probes for structural and dynamic changes of αTS throughout its catalytic cycle, in the absence of the β-subunit. Projection analysis of the chemical shift changes by site-specific amino acid substitutions and ligand titrations indicates that αTS has three important conformational states: ligand-free, glyceraldehyde-3-phosphate-bound(like), and the active states. The amino acid networks within these conformations are different, as suggested by chemical shift correlation analysis. In particular, there are long-range connections, only in the active state, between Ala47, which reports on structural and dynamic changes associated with the general acid/base Glu49, and residues within the β2α2 loop, which contains the catalytically important Asp60 residue. These long-range interactions are likely important for coordinating chemical catalysis. In the free state, but not in the active state, there are connections between the β2α2 and β6α6 loops that likely help to coordinate substrate binding. Changes in the allosteric networks are also accompanied by protein dynamic changes. During catalytic turnover, the protein becomes more rigid on the millisecond timescale and the active-site dynamics are driven to a faster nanosecond timescale.",
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AU - Axe, Jennifer M.

AU - Boehr, David D.

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N2 - The α-subunit of tryptophan synthase (αTS) catalyzes the conversion of indole-3-glycerol phosphate to d-glyceraldehyde-3-phosphate and indole. We propose that allosteric networks intrinsic to αTS are modulated by the binding of the β-subunit to regulate αTS function. Understanding these long-range amino acid networks in αTS thus gives insight into the coordination of the two active sites within TS. In this study, we have used Ala residues as probes for structural and dynamic changes of αTS throughout its catalytic cycle, in the absence of the β-subunit. Projection analysis of the chemical shift changes by site-specific amino acid substitutions and ligand titrations indicates that αTS has three important conformational states: ligand-free, glyceraldehyde-3-phosphate-bound(like), and the active states. The amino acid networks within these conformations are different, as suggested by chemical shift correlation analysis. In particular, there are long-range connections, only in the active state, between Ala47, which reports on structural and dynamic changes associated with the general acid/base Glu49, and residues within the β2α2 loop, which contains the catalytically important Asp60 residue. These long-range interactions are likely important for coordinating chemical catalysis. In the free state, but not in the active state, there are connections between the β2α2 and β6α6 loops that likely help to coordinate substrate binding. Changes in the allosteric networks are also accompanied by protein dynamic changes. During catalytic turnover, the protein becomes more rigid on the millisecond timescale and the active-site dynamics are driven to a faster nanosecond timescale.

AB - The α-subunit of tryptophan synthase (αTS) catalyzes the conversion of indole-3-glycerol phosphate to d-glyceraldehyde-3-phosphate and indole. We propose that allosteric networks intrinsic to αTS are modulated by the binding of the β-subunit to regulate αTS function. Understanding these long-range amino acid networks in αTS thus gives insight into the coordination of the two active sites within TS. In this study, we have used Ala residues as probes for structural and dynamic changes of αTS throughout its catalytic cycle, in the absence of the β-subunit. Projection analysis of the chemical shift changes by site-specific amino acid substitutions and ligand titrations indicates that αTS has three important conformational states: ligand-free, glyceraldehyde-3-phosphate-bound(like), and the active states. The amino acid networks within these conformations are different, as suggested by chemical shift correlation analysis. In particular, there are long-range connections, only in the active state, between Ala47, which reports on structural and dynamic changes associated with the general acid/base Glu49, and residues within the β2α2 loop, which contains the catalytically important Asp60 residue. These long-range interactions are likely important for coordinating chemical catalysis. In the free state, but not in the active state, there are connections between the β2α2 and β6α6 loops that likely help to coordinate substrate binding. Changes in the allosteric networks are also accompanied by protein dynamic changes. During catalytic turnover, the protein becomes more rigid on the millisecond timescale and the active-site dynamics are driven to a faster nanosecond timescale.

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