Hydroxide Once or Twice? A combined Neutron Crystallographic and Quantum Chemical Study of the Hydride Shift in D-Xylose Isomerase

AbstractThe hydride shift mechanism of D-Xylose Isomerase converts D-glucose to D-fructose. In this work, we compute features of a \"hydroxide once\" mechanism for the hydride shift with quantum chemical calculations based on the 3KCO (linear) and 3KCL (cyclic) X-ray/neutron structures. The rigid boundary conditions of the active site \"shoe-box\", together with ionization states and proton orientations, enables large scale electronic structure calculations of the entire active site with greatly reduced configuration sampling. In the reported hydroxide once mechanism, magnesium in the 2A ligation shifts to position 2B, ionizing the O2 proton of D-glucose, which is accepted by ASP-287. In this step a novel stabilization is discovered; the K183/D255 proton toggle, providing a ~10 kcal/mol stabilization through inductive polarization over 5[A]. Then, hydride shifts from glucose-O2 to glucose-O1 (the interconversion) generating hydroxide (once) from the catalytic water. This step is consistent with the observation of hyroxide in structure 3CWH, which we identify as a branch point. From this branch point, we find several routes to the solvent-free regeneration of catalytic water that is strongly exothermic (by ~20 kcal/mol), yielding one additional hydrogen bond more than the starting structure. This non-Michaelis behavior, strongly below the starting cyclic and linear total energy, explains the observed accumulation of hydroxide intermediate - we postulate that forming permissive ionization states, required for cyclization, may be the rate limiting step. In all, we find eight items of experimental correspondence supporting features of the putative hydroxide once mechanism.