TRITIUM ISOTOPE EFFECTS IN ADENOSYLCOBALAMIN-DEPENDENT GLUTAMATE MUTASE - IMPLICATIONS FOR THE MECHANISM

The transfer of tritium between adenosylcobalamin and substrate in the reaction catalyzed by glutamate mutase was examined to investigate th e possibility of a protein-based radical intermediate. There was no ev idence that tritium was transferred to the protein during the reaction , as tritium neither became stably bound to the protein nor exchanged with water. The kinetics of tritium transfer from adenosylcobalamin to 3-methylaspartate was investigated. Both the transfer of tritium to p roduct and the exchange of enzyme-bound and free coenzyme contribute t o the kinetics of tritium loss from adenosylcobalamin. By varying the experimental conditions, the rates of both coenzyme exchange and triti um transfer could be measured. Exchange of adenosylcobalamin with enzy me is very slow, k(off) = 0.01 s(-1), which may reflect a conformation al change in the coenzyme and/or protein involved in forming active ho le enzyme. The rate constants for the loss of tritium from adenosylcob alamin and the appearance of tritium in 3-methylaspartate are much fas ter and very similar, k = 0.67 +/- 0.05 s(-1) and k = 0.50 +/- 0.05 s( -1), respectively, consistent with the transfer of tritium occurring d irectly between coenzyme and substrate. The isotope effect, calculated from the rate constants for tritium transfer, and k(cat), determined for the overall reaction under the same conditions, are between 13.5 a nd 18. These values are typical of primary isotope effects seen for en zymes in which hydrogen transfer is substantially rate limiting. A pro tein radical, therefore, appears unlikely to feature in the mechanism of this enzyme.