Water as a reactant in the first step of triosephosphate isomerase catalysis

The enzyme triosephosphate isomerase (TIM) performs a crucial role in the extraction of energy from glucose, doing so by converting dihydroxyacetone phosphate (DHAP) into glyceraldehyde phosphate, thereby doubling the yield of ATP molecules during glycolysis. The initial step of the mechanism is the seemingly unlikely abstraction of the pro-R methylene hydrogen from C1 by a conserved glutamate (Glu165), an assignment that has been both universally accepted yet a much-studied phenomenon for decades. In this work we introduce an alternative mechanism in which water as a strong general base abstracts the carbon proton acting effectively as hydroxide. We posit that strong electric fields associated with the substrate phosphate promote facile autoionization of water trapped near the phosphate dianion of DHAP and Glu165, an example of substrate assisted catalysis. Classical molecular dynamics simulations assert that the closest water oxygen atom is consistently closer to the pro-R H than the carboxylate oxygen atoms of the accepted base Glu165. Our proposal is further supported by quantum computations that confirm the implausibility of abstraction of the methylene hydrogen by glutamate and the ease with which it is abstracted by hydroxide. The necessity of Glu165 for efficient catalysis is attributed to its crucial involvement in trapping the vital water in an environment of high electric fields which promote ionization far more rapidly than in bulk solvent.