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

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.