Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze a variety of reactions that proceed through organic radical intermediates generated by homolytic fission of coenzyme's unique cobalt-carbon bond. For all the enzymes that have been examined, the homolysis step is kinetically indistinguishable from abstraction of hydrogen from the substrate (or protein), implying that deoxyadenosyl radical is formed only as a fleeting intermediate. To examine how these two steps are coupled together, we have used pre-steady-state, rapid quench techniques to measure the alpha-secondary tritium isotope effect associated with the formation of 5'-deoxyadenosine when the enzyme is reacted with [5'-(3)H]-adenosylcobalamin and L-glutamate. Surprisingly, a large inverse equilibrium isotope effect of 0.72 +/- 0.04 was found for the overall reaction, indicating that the 5'-C-H bonds become significantly stiffer on going from adenosylcobalamin to 5'-deoxyadenosine, even though the 5'-carbon remains formally sp(3) hybridized. The kinetic isotope effect for the formation of 5'-deoxyadenosine was 0.76 +/- 0.02, which suggests a late transition state for the reaction.