Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase.

Glutamate mutase is one of a group of adenosylcobalamin1 (AdoCbl, coenzyme B12) dependent enzymes that catalyze unusual carbon skeleton isomerisations. These rearrangements formally involve a 1,2 hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates (1-6). The initial steps of these reactions involve homolysis of the reactive cobalt-carbon bond of the coenzyme to form cob(II)alamin and 5’-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5-deoxyadenosine and substrate radical (or protein radical in the case of AdoCbl-dependent ribonucleotide reductase). These steps have been studied in some detail for several B12 enzymes and in each case homolysis and hydrogen abstraction are found to be kinetically coupled (7-10), as evidenced by the appearance of a kinetic isotope effect on cobalt-carbon bond homolysis when the enzymes are reacted with deuterated substrates. This observation implies that adenosyl radical can only be present in very low concentrations as a high energy intermediate that does not accumulate on the enzyme. Arguments have been advanced for a formally concerted mechanism for homolysis and hydrogen abstraction (11), although this is now considered to be less likely (12). In many cases these deuterium isotope effects are unusually large, in the range of 40−30, and the most likely explanation for this is that hydrogen transfer involves a large degree of quantum tunneling (13, 14). We have recently described rapid quench experiments (15) to measure the α-secondary tritium isotope effects associated with the formation of 5’-deoxyadensosine under pre-steady state conditions, as illustrated in Figure 1. AdoCbl, tritium-labeled in the 5’-position, was used to measure both the secondary equilibrium and kinetic isotope effects on the formation of 5’-dA. The measurements were made at very short time intervals between 10 and 100 ms so that loss of tritium from the 5’-position was minimal. Both the kinetic and equilibrium isotope effects were found to be large and inverse, kH/kT = 0.76 ± 0.02, KH/KT = 0.72 ± 0.04. These results indicate that the 5’-C-H bonds become significantly stiffer in going from AdoCbl to 5’-dA, even though the 5’-carbon remains formally sp3 hybridized, and are consistent with the hydrogen transfer, as opposed cobalt-carbon bond cleavage, being the slower step. Classically, the large inverse kinetic isotope effect would be interpreted as indicating a late transition state. However, if quantum tunneling is occurring in the hydrogen transfer step, this conclusion cannot be drawn (16). Figure 1 Top: Isomerization reaction catalyzed by glutamate mutase, with the migrating hydrogen atom circled. Bottom: Secondary isotope effects associated with the homolysis of AdoCbl and the formation 5’-dA when hologlutamate mutase is reacted with deuterated ... To investigate whether quantum tunneling is likely to be important in the mechanism of glutamate mutase, we have repeated the secondary α-tritium isotope effect measurements for the formation of 5’-dA using deuterated glutamate to trigger homolysis of AdoCbl. This introduces a primary deuterium isotope effect at the 5’-carbon, which should not change the secondary isotope effect if this arises purely semi-classically (16). However, we observe that the secondary kinetic isotope effect is significantly reduced in magnitude, which implies that the motions of the primary and secondary hydrogen atoms are coupled together in the transition state and that the reaction involves a significant degree of quantum tunneling.