Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (βTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. We show here that substitution of a distant, surface-exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating βTS activity. While stimulation is modest, the substitution also enhances cell growth in a tryptophan-auxotrophic strain of Escherichia coli compared to complementation with wild-type αTS, emphasizing the biological importance of the network. Surface-exposed networks provide new opportunities in allosteric drug design and protein engineering, and hint at potential information conduits through which the functions of a metabolon or even larger proteome might be coordinated and regulated.

A general framework by which dynamic interactions within a protein will promote the necessary series of structural changes, or “conformational cycle,” required for function is proposed. It is suggested that the free‐energy landscape of a protein is biased toward this conformational cycle. Fluctuations into higher energy, although thermally accessible, conformations drive the conformational cycle forward. The amino acid interaction network is defined as those intraprotein interactions that contribute most to the free‐energy landscape. Some network connections are consistent in every structural state, while others periodically change their interaction strength according to the conformational cycle. It is reviewed here that structural transitions change these periodic network connections, which then predisposes the protein toward the next set of network changes, and hence the next structural change. These concepts are illustrated by recent work on tryptophan synthase. Disruption of these dynamic connections may lead to aberrant protein function and disease states.

Tryptophan synthase (TS) is a α2β2 dimeric enzyme that catalyzes the final two reactions in the production of tryptophan. The product of the alpha subunit (αTS), indole, is channeled to the beta subunit (βTS) where it is then condensed with serine to form tryptophan. TS is a valuable model system for understanding enzyme allostery, and specifically enzyme‐enzyme communication. We previously identified amino acid interaction networks in αTS using NMR‐based methods. We proposed that these network residues play an important role in catalysis and communication between the two subunits. Based on previous studies, αTS residue Ala198 was identified as a residue of interest. Ala198 is a surface‐exposed, network residue that is dynamic on the ms‐μs timescale, according to our previous NMR studies. To better understand the networks, we characterized the A198W variant by performing cell‐based assays, kinetic assays, molecular dynamics (MD) simulations, and NMR experiments. We found that E.coli cells expressing the A198W variant had a faster growth rate in minimal medium compared to cells expressing the wild‐type αTS. Our kinetic studies on purified TS indicated that the A198W variant had little effect on αTS activity, but increased the activity of the TS complex (i.e. from initial indole‐3‐glycerol phosphate substrate to final tryptophan product), perhaps due to an enhancement of indole channeling. MD simulations indicated that the A198W substitution led to structural dynamic changes that propagated throughout αTS, including several key residues at the α/β binding interface. The MD simulations were consistent with the indole channel being more open, more often, which could help explain the increase in TS activity in vitro and in vivo. NMR experiments also indicated that the A198W substitution induced structural dynamic changes throughout αTS, consistent with the MD simulations. Despite Ala198 being distal from both the active site of αTS (27Å) and the α/β binding interface (30Å), substitutions at this position result in changes to catalytic efficiency and the TS complex dynamics. These findings underscore the importance of network residues on enzyme dynamics, allostery, and subunit communication. Engineering of surface‐exposed network residues represents one way of controlling enzyme activity. 6.5.0 Support or Funding Information 6.5.06.5.0