Characterization of an allylic analogue of the 5 '-deoxyadenosyl radical: An intermediate in the reaction of lysine 2,3-aminomutase

An allylic analogue of the 5'-deoxyadenosyl radical has been characterized at the active site of lysine 2,3-aminomutase (LAM) by electron paramagnetic resonance (EPR) spectroscopy. The anhydroadenosyl radical, 5'-deoxy-3',4'- anhydroadenosine-5'-yl, is a surrogate of the less stable 5'-deoxyadenosyl radical, which has never been observed but has been postulated to be a radi cal intermediate in the catalytic cycles of a number of enzymes, An earlier communication [Magnusson, O.Th., Reed, G. H., and Frey, P. A. (1999) J. Am . Chem. Sec. 121, 9764-9765] included the initial spectroscopic identificat ion at 77 K of the radical, which is formed upon replacement of S-adenosylm ethionine by S-3',4'-anhydroadenosylmethionine as a coenzyme for LAM. The e lectron paramagnetic resonance spectrum of the radical changes dramatically between 77 and 4.5 K. This unusual temperature dependence is attributed to a spin-spin interaction between the radical and thermally populated, highe r spin states of the [4Fe-4S](+2) center, which is diamagnetic at 4.5 K, Th e EPR spectra of the radical at 4.5 K have been analyzed using isotopic sub stitutions and simulations. Analysis of the nuclear hyperfine splitting sho ws that the unpaired spin is distributed equally between C5'- and C3'- as e xpected for an allylic radical. Hyperfine splitting from the beta -proton a t C-2'(H) shows that the dihedral angle to the p(z)-orbital at C-3' is appr oximately 37 degrees. This conformation is in good agreement with a structu ral model of the radical. The rate of formation of the allylic radical show s that it is kinetically competent as an intermediate. Measurements of H-2 kinetic isotope effects indicate that with lysine as the substrate, the rat e-limiting steps follow initial reductive cleavage of the coenzyme analogue .