Hw. Chih et Eng. Marsh, Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase, BIOCHEM, 40(43), 2001, pp. 13060-13067
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.