The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend temperature-dependent hydrogen deuterium exchange from apo-enzyme structures to protein-ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different hydrogen deuterium exchange-mass spectrometry properties for the two ligands, as expected from their 10⁶-fold differences in strength of binding. By contrast, analogous patterns for temperature-dependent hydrogen deuterium exchange mass spectrometry emerge in the presence of both 1-deaza-adenosine and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended temperature-dependent hydrogen deuterium exchange to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility versus site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient 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 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.

Murine adenosine deaminase (mADA) is a prototypic system for studying the thermal activation of active site chemistry within the TIM barrel family of enzyme reactions. Previous temperature-dependent hydrogen deuterium exchange studies under various conditions have identified interconnected thermal networks for heat transfer from opposing protein-solvent interfaces to active site residues in mADA. One of these interfaces contains a solvent exposed helix-loop-helix moiety that presents the hydrophobic face of its long α-helix to the backside of bound substrate. Herein we pursue the time and temperature dependence of solvation dynamics at the surface of mADA, for comparison to established kinetic parameters that represent active site chemistry. We first created a modified protein devoid of native tryptophans with close to native kinetic behavior. Single site-specific tryptophan mutants were back inserted into each of the four positions where native tryptophans reside. Measurements of nanosecond fluorescence relaxation lifetimes and Stokes shift decays, that reflect time dependent environmental reoroganization around the photo-excited state of Trp*, display minimal temperature dependences. These regions serve as controls for the behavior of a new single tryptophan inserted into a solvent exposed region near the helix-loop-helix moiety located behind the bound substrate, Lys54Trp. This installed Trp displays a significantly elevated value for Ea(kStokesshift) ; further, when Phe61 within the long helix positioned behind bound substrate is replaced by a series of aliphatic hydrophobic side chains, the trends in Ea(kStokesshift) mirror the earlier reported impact of the same series of function-altering hydrophobic side chains on the activation energy of catalysis, Ea(kcat) .The reported experimental findings implicate a solvent initiated and rapid (>ns) protein restructuring that contributes to the enthalpic activation barrier to catalysis in mADA.