Kinetic Isolation and Characterization of the Radical Rearrangement Step in Coenzyme B12-Dependent Ethanolamine Ammonia-lyase

摘要

The transient decay reaction kinetics of the 1,1,2,2-2H₄-aminoethanol generated CoII−substrate radical pair catalytic intermediate in ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been measured by using time-resolved, full-spectrum X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy in frozen aqueous solution over the temperature range of 190−207 K. The decay reaction involves sequential passage through the rearrangement step [substrate radical → product radical] and the step [product radical → diamagnetic product] that involves hydrogen atom transfer (HT) from carbon C5′ of the adenosine moiety of the cofactor to the product radical C2 center. As found for the 1H−substrate radical [Zhu, C.; Warncke, K. Biophys. J. 2008, 95, 5890], the decay kinetics for the 2H−substrate radical over 190−207 K represent two noninteracting populations (fast decay population: normalized amplitude = 0.44 ± 0.07; observed rate constant, k_obs,f = 5.3 × 10−5−1.1 × 10−3 s−1; slow decay population: k_obs,s = 6.1 × 10−6−2.9 × 10−4 s−1). The 1H/2H isotope effects (IE) for the fast and slow decay reactions are 1.4 ± 0.2 and 0.79 ± 0.11, respectively. The IE on the fast phase is uniform over the temperature interval, and the value is consistent with an α-secondary hydrogen kinetic IE, which arises from changes in the force constants of the C−H bonds in the substrate radical structure, upon passing from the substrate radical state to the rearrangement transition state. Therefore, we propose that k_obs,f represents the rate constant for the radical rearrangement and that this step is the rate-determining step in substrate radical decay. The Arrhenius activation energy for the 1H-substrate radical rearrangement (13.5 ± 0.4 kcal/mol) is consistent with values from quantum chemical calculations performed on simple models. The results show that the core, radical rearrangement reaction is culled from the catalytic cycle in the low-temperature system, thus establishing the system for detailed transient kinetic and spectroscopic analysis of protein structural and dynamic contributions to EAL catalysis.