Abstract
Orotidine 5'-monophosphate decarboxylase (OMPDC) is one of the most efficient enzyme systems studied, enhancing the decarboxylation of OMP to uridine 5'-monophosphate (UMP) by ca. 17 orders of magnitude, primarily by reducing the enthalpy of activation by ca. 28 kcal/mol. Despite a substantial reduction in activation enthalpy, OMPDC requires 15 kcal/mol of activation energy post-ES complex formation. This study investigates the physical basis of how thermal energy from solvent collisions is directed into the active site of enzyme to enable efficient thermal activation of the reaction. Comparative study of temperature-dependent hydrogen-deuterium exchange mass spectrometry (TDHDX) for WT and mutant forms of enzymes has recently been shown to uncover site specific protein networks for thermal energy transfer from solvent to enzyme active sites. In this study, we interrogate region-specific changes in the enthalpic barrier for local protein flexibility using a native OMPDC from Methanothermobacter thermautotrophicus (Mt-OMPDC) and a single site variant (Leu123Ala) that alters the activation enthalpy for catalytic turnover. The data obtained implicate four spatially resolved, thermally sensitive networks that originate at different protein/solvent interfaces and terminate at sites surrounding the substrate near the substrate phosphate-binding region (R203), the substrate- ribose binding region (K42), and a reaction enhancing loop5 (S127). These are proposed to act synergistically, transiently optimizing the position and electrostatics of the reactive carboxylate of the substrate to facilitate activated complex formation. The uncovered complexity of thermal activation networks in Mt-OMPDC distinguishes this enzyme from other members of the TIM barrel family previously investigated by TDHDX. The new findings extend the essential role of protein scaffold dynamics in orchestrating enzyme activity, with broad implications for the design of highly efficient biocatalysts.