The role of the active site glutamate in the rearrangement of glutamate to 3-methylaspartate catalyzed by adenosylcobalamin-dependent glutamate mutase

AbstractBackground: Adenosylcobalamin (coenzyme B12)-dependent enzymes catalyze a variety of chemically difficult reactions that proceed through the generation of free radical intermediates. A long-standing question is how proteins stabilize what are normally regarded as highly reactive organic radicals and direct them towards productive reactions. In glutamate mutase the carboxylate of Glu171 hydrogen bonds with the amino group of the substrate. We have investigated the role of this residue in the enzyme mechanism.Results: Several sterically and functionally conservative mutations were introduced at position 171. In the most impaired mutant, Glu171Gln, kcat is reduced 50-fold, although the Km for glutamate is little affected. In the wild-type enzyme activity was pH-dependent and the acidic limb of the activity curve titrated with an apparent pKa of 6.6 on Vmax, whereas for the sluggish Glu171Gln mutant activity is independent of pH. The steady state deuterium kinetic isotope effect is reduced in the mutant enzyme, but the steady state concentration of free radical species on the enzyme (as measured by the steady state concentration of cob(II)alamin) is unaffected by the mutation.Conclusions: The properties of the mutant proteins are consistent with the hypothesis that Glu171 acts as a general base that serves to deprotonate the amino group of the substrate during catalysis. Deprotonation is expected to facilitate the formation of the glycyl radical intermediate formed during the inter-conversion of substrate and product radicals, but to have little effect on the stability of product or substrate radicals themselves