Entropic origin for catalysis of cobalt-carbon bond cleavage in coenzyme B12 (adenosylcobalamin) in ethanolamine ammonia-lyase

The formation of the CoII-substrate radical pair catalytic intermediate in coenzyme B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been studied in a 41% (v:v) DMSO/water cryosolvent system in the temperature range of 230-250 K by using X-band electron paramagnetic resonance (EPR) spectroscopy. For the first time, a stable (>4 hr at 230 K) ternary complex of enzyme, coenzyme and substrate is formed in a coenzyme B12 -dependent enzyme, which allows temperature-step initiation of the reaction to form the CoII-substrate radical pair, and monitoring by time-resolved, full-spectrum EPR spectroscopy. A three-state (intact AdoCbl, substrate bound initial state; CoII-5'-deoxyadenosyl radical pair intermediate state; CoII-substrate radical pair state), two-step mechanism is used to treat the CoII-substrate radical pair formation reaction kinetics and equilibria with explicit consideration of the intermediate radical pair state. By using this model, the absence of an EPR-detectable intermediate yields a limit of >3.3 kcal/mol for the free energy of the CoII-5'-deoxyadenosyl radical pair relative to the ternary complex. The free energy difference between the CoII -substrate radical pair state and the initial state is approximately 0 kcal/mol for 230-250 K, and has an extrapolated value of -2.6 kcal/mol at 298 K. The absence of a substrate hydrogen isotope effect on the rate of Co II-substrate radical pair formation indicates that the Co-C bond cleavage is rate determining at 230-250 K. This allows the first-time determination, by using Eyring analysis, of the Co-C bond cleavage activation enthalpy and entropy in a coenzyme B12-dependent enzyme. The large, 16 kcal/mol decrease in the activation free energy for Co-C bond cleavage in EAL relative to solution is contributed almost entirely by a large, positive activation entropy. The activation parameters offer a quantitative description for the coupling between the Co-C bond cleavage and hydrogen transfer steps in EAL. The UV-visible spectroscopy is used to obtain optical spectrum from the ternary complex. The result indicates that the ground state destabilization (enthalpic strain) is not a significant catalysis contribution, and supports the findings from the EPR studies. These results provide a new paradigm for catalysis of Co-C bond cleavage in coenzyme B12-dependent enzymes.