Dynamically Interacting Protein Networks Provide a Mechanism to Overcome the Enormous Intrinsic Barrier to Orotidine 5′-Monophosphate Decarboxylation

Abstract

Orotidine 5′-monophosphate decarboxylase (OMPDC) is among the most efficient enzymes known, accelerating the decarboxylation of the OMP by ∼17 orders of magnitude, primarily by lowering the enthalpy of activation by ∼28 kcal/mol. Despite this feature, OMPDC from Methanothermobacter thermautotrophicus requires ∼15 kcal/mol of activation energy following ES complex formation. This study applies temperature-dependent hydrogen–deuterium exchange mass spectrometry (TDHDX) to detect site-specific thermal protein networks that channel energy from solvent collisions to the active site. Comparative TDHDX of native OMPDC and a single-site variant (Leu123Ala) that alters the activation enthalpy for catalytic turnover reveals region-specific changes in protein flexibility, connecting local scaffold unfolding enthalpy to the activation barrier of catalysis. The data implicate four spatially resolved, thermally sensitive networks that originate at distinct protein–solvent interfaces and converge near the substrate phosphate-binding region (R203), the ribose-binding region (K42), and a catalytic loop (S127). These networks are proposed to act synergistically to optimize substrate positioning and active site electrostatics for the activated complex formation. The complexity of the identified thermal activation pathways distinguishes Mt-OMPDC from other TIM barrel enzymes previously studied by TDHDX. The findings highlight the essential role of scaffold dynamics in enzyme function with broad implications for designing efficient biocatalysts.

This study uncovers multiple thermal energy transfer pathways in OMPDC, highlighting how site-specific protein dynamics facilitate substrate positioning and electrostatics for C−C bond cleavage.