RESUMO
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 ( k Stokes shift ) ; 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 ( k Stokes shift ) mirror the earlier reported impact of the same series of function-altering hydrophobic side chains on the activation energy of catalysis, Ea ( k cat ) .The reported experimental findings implicate a solvent initiated and rapid (>ns) protein restructuring that contributes to the enthalpic activation barrier to catalysis in mADA.