Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase

Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and prod uct in the reaction catalyzed by glutamate mutase. The isotope partitions, between glutamate and methylaspartate in nearly 1:1 ratio, regardless of th e direction in which the overall reaction is proceeding. This is consistent with a free-energy profile in which the interconversion of the intermediat e glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product. Initial vel ocity measurements have been used to measure the tritium isotope affects fo r the transfer of tritium from adenosylcobalamin to product in each directi on. The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate. The large magnitude of these isotope effects m akes it likely that the rate-determining step may be altered by the substit ution of tritium for hydrogen in the reaction. The results of these experim ents are compared with previous isotope effect measurements made on other a denosylcobalamin-dependent enzymes.