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

Abstract

The transient decay reaction kinetics of the 1,1,2,2-(2)H(4)-aminoethanol generated Co(II)-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 (1)H-substrate radical [Zhu, C.; Warncke, K. Biophys. J. 2008, 95, 5890], the decay kinetics for the (2)H-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 x 10(-5)-1.1 x 10(-3) s(-1); slow decay population: k(obs,s) = 6.1 x 10(-6)-2.9 x 10(-4) s(-1)). The (1)H/(2)H 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 alpha-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 (1)H-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.