Lysine 2,3-aminomutase and trans-4,5-dehydrolysine: Characterization of anallylic analogue of a substrate-based radical in the catalytic mechanism?

An analogue of lysine, trans-4,5-dehydro-L-lysine (trans-4,5-dehydrolysine) , is a potent inhibitor of lysine 2,3-aminomutase from Clostridium subtermi nale SB4 that competes with L-lysine for binding to the active site. Inclus ion of trans-4,5-dehydrolysine with activated enzyme and the coenzymes pyri doxal-5'-phosphate and S-adenosylmethionine, followed by freezing at 77 K, produces an intense signal in the electron paramagnetic resonance (EPR) spe ctrum at g 2.0, which is characteristic of an organic radical. A series of deuterated and N-15-labeled samples of trans-4,5-dehydrolysine were synthes ized and used to generate the EPR signal. Substitution of deuterium for hyd rogen at C2, C3, C4, C5, and C6 of trans-4,5-dehydrolysine led to significa nt simplifications and narrowing of the EPR signal, showing that the unpair ed electron was located on the carbon skeleton of 4,5-trans-4,5-dehydrolysi ne. The hyperfine splitting pattern is simplified by use of 4,5-dehydro[3,3 -H-2(2)]lysine or 4,5-dehydro[4,5-H-2(2)]lysine, and it is dramatically sim plified with 4,5-dehydro-[3,3,4,5,6,6-H-2(6)]lysine, Spectral simulations s how that the EPR signal arises from the allylic radical resulting from the abstraction of a hydrogen atom from C3 of trans-4,5-dehydrolysine This radi cal is an allylic analogue of the substrate-related radical in the rearrang ement mechanism postulated for this enzyme. The rate constant for formation of the 4,5-dehydrolysyl radical (2 min(-1)) matches that for the decrease in the concentraion of [4Fe-4S](+), showing that the two processes are coup led. The cleavage of S-adenosylmethionine to 5'-deoxyadenosine and methioni ne takes place with a rate constant of approximately 5 min(-1). These kinet ic correlations support the hypothesis that radical formation results from a reversible reaction between [4Fe-4S](+) and S-adenosylmethionine at the a ctive site to form [4Fe-4S](2+), the 5'-deoxyadenosyl radical, and methioni ne as intermediates.