title: Characterization of an allylic analogue of the 5'-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase.
abstract: 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 radical 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. Soc. 121, 9764-9765] included the initial spectroscopic identification at 77 K of the radical, which is formed upon replacement of S-adenosylmethionine by S-3',4'-anhydroadenosylmethionine as a coenzyme for LAM. The electron 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, higher spin states of the [4Fe-4S]+2 center, which is diamagnetic at 4.5 K. The EPR spectra of the radical at 4.5 K have been analyzed using isotopic substitutions and simulations. Analysis of the nuclear hyperfine splitting shows that the unpaired spin is distributed equally between C5'- and C3'- as expected for an allylic radical. Hyperfine splitting from the beta-proton at C-2'(H) shows that the dihedral angle to the p(z)-orbital at C-3' is approximately 37 degrees. This conformation is in good agreement with a structural model of the radical. The rate of formation of the allylic radical shows that it is kinetically competent as an intermediate. Measurements of 2H kinetic isotope effects indicate that with lysine as the substrate, the rate-limiting steps follow initial reductive cleavage of the coenzyme analogue.
authors: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
authors: O T
authors: OT
authors: Magnusson
authors: G H
authors: GH
authors: Reed
authors: P A
authors: PA
authors: Frey
mesh_terms: D000498:Allyl Compounds / Q000138:chemical synthesis*; D002247:Carbon Isotopes; D003013:Clostridium / Q000201:enzymology; D003067:Coenzymes / Q000138:chemical synthesis; D003839:Deoxyadenosines / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D005609:Free Radicals / Q000737:chemistry; D019751:Intramolecular Transferases / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000138:chemical synthesis; D013696:Temperature
chemical_list: D000498:Allyl Compounds; D002247:Carbon Isotopes; D003067:Coenzymes; D003839:Deoxyadenosines; D005609:Free Radicals; C033714:5'-deoxyadenosine; D012436:S-Adenosylmethionine; D003903:Deuterium; D019751:Intramolecular Transferases; C021518:lysine 2,3-aminomutase