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

Proteins are intrinsically flexible
macromolecules that undergo
internal motions with time scales spanning femtoseconds to milliseconds.
These fluctuations are implicated in the optimization of reaction
barriers for enzyme catalyzed reactions. Time, temperature, and mutation
dependent hydrogen–deuterium exchange coupled to mass spectrometry
(HDX-MS) has been previously employed to identify spatially resolved,
catalysis-linked dynamical regions of enzymes. 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 = 7.5 kcal/mol) and the wild type (WT)
mADA (Ea = 5.0 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 kcat 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.