Severing of a hydrogen bond disrupts amino acid networks in the catalytically active state of the alpha subunit of tryptophan synthase

Abstract

Conformational changes in the b2a2 and b6a6 loops in the alpha subunit of tryptophan synthase (aTS) are important for enzyme catalysis and coordinating substrate channeling with the beta subunit (bTS). It was previously shown that disrupting the hydrogen bond interactions between these loops through the T183V substitution on the b6a6 loop decreases catalytic efficiency and impairs substrate channeling. Results presented here also indicate that the T183V substitution decreases catalytic efficiency in Escherchia coli aTS in the absence of the bTS subunit. Nuclear magnetic resonance (NMR) experiments indicate that the T183V substitution leads to local changes in the structural dynamics of the b2a2 and b6a6 loops. We have also used NMR chemical shift covariance analyses (CHESCA) to map amino acid networks in the presence and absence of the T183V substitution. Under conditions of active catalytic turnover, the T183V substitution disrupts long-range networks connecting the catalytic residue Glu49 to the aTS-bTS binding interface, which might be important in the coordination of catalytic activities in the tryptophan synthase complex. The approach that we have developed here will likely find general utility in understanding long-range impacts on protein structure and dynamics of amino acid substitutions generated through protein engineering and directed evolution approaches, and provide insight into disease and drug-resistance mutations.

Engineered control of enzyme structural dynamics and function

“…Our analysis revealed that there are two networks of residues [colored orange and blue in Fig. (B)], and the residues involved in these networks change depending on what is bound to the protein . Intriguingly, Glu49, a residue directly involved in catalysis, belongs to different networks when comparing the apo resting state and when the enzyme is actively turning over substrate/products (i.e., the working state) .…”

“…These networks may also be important for how αTS communicates and coordinates channeling of its product indole into the active site of the beta subunit of tryptophan synthase (βTS). An amino acid substitution known to disrupt direct channeling of indole from the α‐ to β‐active sites also disrupts networks that might transmit structural dynamic information from the α‐active site to the α/β binding interface …”

“…3(B)], and the residues involved in these networks change depending on what is bound to the protein. [110][111][112] Intriguingly, Glu49, a residue directly involved in catalysis, belongs to different networks when comparing the apo resting state and when the enzyme is actively turning over substrate/products (i.e., the working state). 111 Molecular dynamics (MD) simulations also indicated that the motions of Glu49 are correlated to different sets of residues in these states.…”

Solution NMR Spectroscopy for the Study of Enzyme Allostery

“…Other NMR studies on the enzyme tryptophan synthase have identified long-range allosteric pathways and have mechanistically investigated these pathways by mutations that disrupt critical interactions. 77,78 …”

Investigation of Structural Dynamics of Enzymes and Protonation States of Substrates Using Computational Tools

“…NMR chemical shift covariance analyses (CHESCA) and MD simulations are powerful tools to detect such amino acid networks when substrates or products are absent (resting state) and present (working state) in the active site [95–97]. We investigated TRPS in its resting and working states to identify the correlation networks from a cluster of residues.…”