(719b) Hydrogen–Deuterium Exchange within Adenosine Deaminase, a TIM Barrel Hydrolase, Identifies Networks for Thermal Activation of Catalysis

The classical, textbook description of the origins of enzyme catalysis has been largely derived from static models of protein structures. However, proteins in solution are in constant motion, a result of thermally activated fluctuations that span a wide range of distances and time scales. The different classes of protein dynamics include large scale conformational changes, domain shifts and local atomic vibrations that are associated with time scales ranging from femtoseconds to seconds. A major, ongoing challenge is the design of experimental approaches that are capable of linking rapid and transient protein motions to enzymatic activity. Recently, temperature-dependent hydrogen-deuterium exchange mass spectrometry (TDHDX-MS) emerged as a broadly applicable experimental probe that provides a three-dimensional map of protein thermal flexibility that can be correlated to enzyme function.

We now extend this technique to pursue the correlation of protein flexibility and chemical reactivity within the diverse and widespread TIM barrel proteins, targeting murine adenosine deaminase (mADA) that catalyzes the irreversible deamination of adenosine to inosine and ammonia. Following a structure–function analysis of rate and activation energy for a series of mutations at a second sphere phenylalanine positioned in proximity to the bound substrate, the catalytically impaired Phe61Ala with an elevated activation energy (Ea = 17.5 kcal/mol) and the wild type (WT) mADA (Ea = 11.0 kcal/mol) were selected for HDX-MS experiments. The rate constants and activation energies of HDX for peptide segments are quantified and used to assess mutation-dependent changes in local and distal motions. Analyses reveal that approximately 50% of the protein sequence of Phe61Ala displays significant changes in the temperature dependence of HDX behaviors, with the dominant change being an increase in protein flexibility. Utilizing Phe61Ile, which displays the same activation energy for k_cat as WT, as a control, we were able to further refine the HDX analysis, highlighting the regions of mADA that are altered in a functionally relevant manner. A map is constructed that illustrates the regions of protein that are proposed to be essential for the thermal optimization of active site configurations that dominate reaction barrier crossings in the native enzyme.

The revealed thermal networks explored within this study offer promising insights for targeted protein engineering and de novo design endeavors where protein structural dynamics play a key role in facilitating enzyme catalysis.