Protein Configurational States Guide Radical Rearrangement Catalysis in Ethanolamine Ammonia-Lyase

Abstract

The adenosylcobalamin- (coenzyme B12) dependent ethanolamine ammonia-lyase (EAL) plays a key role in aminoethanol metabolism, associated with microbiome homeostasis and Salmonella- and Escherichia coli-induced disease conditions in the human gut. To gain molecular insight into these processes toward development of potential therapeutic targets, reactions of the cryotrapped (S)-2-aminopropanol substrate radical EAL from Salmonella typhimurium are addressed over a temperature (T) range of 220–250 K by using T-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy. The observed substrate radical reaction kinetics are characterized by two pairs of biexponential processes: native decay to diamagnetic products and growth of a non-native radical species and Co(II) in cobalamin. The multicomponent low-T kinetics are simulated by using a minimal model, in which the substrate-radical macrostate, S⋅, is partitioned by a free-energy barrier into two sequential microstates: 1) S1⋅, a relatively high-entropy/high-enthalpy microstate with a protein configuration that captures the nascent substrate radical in the terminal step of radical-pair separation; and 2) S2⋅, a relatively low-enthalpy/low-entropy microstate with a protein configuration that enables the rearrangement reaction. The non-native, destructive reaction of S1⋅ at T ≤ 250 K is caused by a prolonged lifetime in the substrate-radical capture state. Monotonic S⋅ decay over 278–300 K indicates that the free-energy barrier to S1⋅ and S2⋅ interconversion is latent at physiological T-values. Overall, the low-temperature studies reveal two protein-configuration microstates and connecting protein-configurational transitions that specialize the S⋅ macrostate for the dual functional roles of radical capture and rearrangement enabling. The identification of new, to our knowledge, intermediate states and specific protein-fluctuation contributions to the reaction coordinate represent an advance toward development of novel therapeutic targets in EAL.