Intrinsic deuterium kinetic isotope effects in glutamate mutase measured by an intramolecular competition experiment.

Kinetic isotope effects (KIEs) provide a powerful tool to interrogate transition states of both enzymic and non-enzymic reactions, provided that one can measure the intrinsic KIE on the chemical step of interest, that is, the isotope effect undiminished by other isotope-insensitive steps that may contribute to the overall rate of reaction. For small molecules reacting in solution the KIE measured usually represents the intrinsic value; however, for enzymes this is seldom the case. Here we report the first measurement of the intrinsic KIE in an adenosylcobalamin enzyme (AdoCbl, coenzyme B12) for hydrogen atom transfer from substrate to coenzyme, which is a key step in the mechanism of this class of enzymes. For the B12 enzyme glutamate mutase the intrinsic deuterium KIE for hydrogen transfer from the substrate, (2S,3S)-3-methylaspartate, to 5’-deoxyadensosine is 4.1. This value is well within the semiclassical limit for a deuterium isotope effect and is much smaller than the anomalously large KIEs previously measured in other B12 enzymes and non-enzymatic model reactions, which were attributed to extensive hydrogen tunneling. Glutamate mutase one is of a group of AdoCbl-dependent enzymes that catalyze unusual isomerization reactions that formally involve a 1,2 hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates (Scheme 1). Radicals are generated by homolysis of the reactive cobalt–carbon bond of the coenzyme to form cob(II)alamin, a cobalt(II) intermediate, and the 5’-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5’-deoxyadenosine and the substrate radical. The substrate radical next undergoes rearrangement to give the product radical, which is then quenched by hydrogen transfer from 5’deoxyadenosine to give the product and regenerate the 5’deoxyadenosyl radical. Finally, recombination of the adenosyl radical and cob(II)alamin to reform the coenzyme completes the catalytic cycle. Our interest in the mechanisms by which enzymes generate free radicals, as exemplified by dependent glutamate mutase, led us to undertake an extensive set of KIE measurements to examine how hydrogen abstraction from the substrate and coenzyme homolysis are coupled together. KIE measurements using deuteriumand tritium-labeled substrates and coenzyme have proved especially informative probes of the key steps of Co C bond homolysis and hydrogen atom abstraction from substrate. Pre-steady-state measurements on a number of enzymes have shown that hydrogen abstraction is kinetically coupled to Co C bond homolysis, 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 the 5’-dA radical is a highenergy intermediate that only has a fleeting existence. Furthermore, the KIEs reported for several AdoCbl enzymes are extremely large (ranging from 10 to 50), which has generally been attributed to hydrogen tunneling. In particular, extensive hydrogen tunneling in methylmalonyl-CoA mutase has been deduced from the temperature dependence of the deuterium isotope effect on hydrogen transfer and Co C bond homolysis. The KIEs discussed above were all measured indirectly by UV/Vis stopped-flow spectroscopy, using the spectroscopic changes associated with Co C bond homolysis as a convenient reporter of the kinetics. Although we originally reported a very large primary deuterium isotope effect, suggestive of tunneling, for the formation of cob(II)alamin and 5’-dA upon reaction of hologlutamate mutase with deuterated substrates (kH/kD= 28 with glutamate and 35 with methylaspartate), [11]