Native and nonnative reactions in ethanolamine ammonia-lyase are actuated by different dynamics

We address the contribution of select classes of solvent-coupled configurational fluctuations to the complex choreography involved in configurational and chemical steps in an enzyme by comparing native and nonnative reactions conducted at different protein internal sites. The low temperature, first-order kinetics of covalent bond rearrangement of the cryotrapped substrate radical in coenzyme B₁₂-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica display a kink, or increase in slope, of the Arrhenius plot with decreasing temperature. The event is associated with quenching of a select class of reaction-actuating collective fluctuations in the protein hydration layer. For comparison, a nonnative, radical reaction of the protein interior cysteine sulfhydryl group with hydrogen peroxide (H₂O₂) is introduced by cryotrapping EAL in an aqueous H₂O₂ eutectic system. The low-temperature aqueous H₂O₂ protein hydration and mesodomain solvent phases surrounding cryotrapped EAL are characterized by using TEMPOL spin probe electron paramagnetic resonance spectroscopy, including a freezing transition of the eutectic phase that orders the protein hydration layer. Kinetics of the cysteine-H₂O₂ reaction in the EAL protein interior are monitored by DEPMPO spin trapping of hydroxyl radical product. In contrast to the native reaction, the linear Arrhenius relation for the nonnative cysteine-H₂O₂ reaction is maintained through the solvent-protein ordering transition. The nonnative reaction is coupled to the generic local, incremental fluctuations that are intrinsic to globular proteins. The comparative approach supports the proposal that select coupled solvent-protein configurational fluctuations actuate the native reaction, and suggests that select dynamical coupling contributes to the degree of catalysis in enzymes.