Isotope Effects for Deuterium Transfer between Substrate and Coenzyme in Adenosylcobalamin-Dependent Glutamate Mutase^†

A key step in the mechanism of all adenosylcobalamin-dependent enzymes is the abstraction of a hydrogen atom from the substrate by a 5‘-deoxyadenosyl radical generated by homolytic fission of the coenzyme cobalt−carbon bond. We have investigated the isotope effects associated with this process for glutamate mutase reacting with deuterated glutamate. The kinetics of deuterium incorporation into 5‘-deoxyadenosine (5‘-dA) during the reaction were followed by rapid chemical quench, using HPLC and electrospray mass spectrometry to analyze the 5‘-dA formed. The kinetics of 5‘-dA formation are biphasic, comprising a rapid phase k_app = 37 ± 3 s^-1 and a slower phase k_app = 0.9 ± 0.4 s^-1. The mass spectral data clearly show that the faster phase is associated with the formation of monodeuterated 5‘-dA whereas the slower phase is associated with the incorporation of a second and then a third deuterium into 5‘-dA. This observation implies that a large inverse equilibrium secondary isotope effect is associated with the formation of 5‘-dA from adenosylcobalamin. The primary deuterium kinetic isotope effects on V and V/K for the formation of 5‘-dA were determined from time-based and competition experiments. ^DV = 2.4 ± 0.4 whereas ^D(V/K) = 10 ± 0.4, implying that an isotopically insensitive step is partially rate-determining. The additional data provided by these experiments cause us to revise our interpretation of earlier UV−visible stopped-flow kinetic measurements of AdoCbl homolysis obtained with deuterated substrates.