Triosephosphate isomerase is a “perfect” enzyme, catalyzing the reversible isomerization of D-glyceraldehyde phosphate to dihydroxyacetone phosphate with partially diffusion-limited character. While the general mechanism of this enzyme has been well studied, the mode of operation of the active site’s hydrophobic clamp remained a puzzle. Our empirical valence bond simulations of the wild-type enzyme and mutations in the hydrophobic clamp revealed that the clamp minimizes the free energy for initial substrate deprotonation. The breakdown of the ground- vs. transition-state effects in our calculations suggests that the driving force for the reaction is predominantly expressed at the transition state for the deprotonation. Following from this, we used a combination of the GPU-enabled long-timescale molecular dynamics simulations coupled with Markov state modeling and various enhanced-sampling techniques in order to probe the general dynamics of a key active site loop. Our detailed computational analysis suggested this loop does not exhibit a simple “open and shut” dynamics, i.e., a rigid body motion around two hinges, as previously proposed in the literature. Rather, it samples a variety of open-state conformations interconnected with low energy barriers, while in the closed state it maintains the proper architecture needed for the efficient catalysis of the chemical step.

Legend of a perfect enzyme: Active site architecture and loop dynamics in triosephosphate isomerase

256th National Meeting (2018)

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