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 subterminale SB4 that competes with L-lysine for binding to the active site. Inclusion of trans-4,5-dehydrolysine with activated enzyme and the coenzymes pyridoxal-5'-phosphate and S-adenosylmethionine, followed by freezing at 77 K, produces an intense signal in the electron paramagnetic resonance (EPR) spectrum at g 2.0, which is characteristic of an organic radical. A series of deuterated and 15N-labeled samples of trans-4,5-dehydrolysine were synthesized and used to generate the EPR signal. Substitution of deuterium for hydrogen at C2, C3, C4, C5, and C6 of trans-4,5-dehydrolysine led to significant simplifications and narrowing of the EPR signal, showing that the unpaired electron was located on the carbon skeleton of 4,5-trans-4,5-dehydrolysine. The hyperfine splitting pattern is simplified by use of 4,5-dehydro[3,3-2H2]lysine or 4,5-dehydro[4,5-2H2]lysine, and it is dramatically simplified with 4,5-dehydro-[3,3,4,5,6,6-2H6]lysine. Spectral simulations show 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 radical is an allylic analogue of the substrate-related radical in the rearrangement 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 coupled. The cleavage of S-adenosylmethionine to 5'-deoxyadenosine and methionine takes place with a rate constant of approximately 5 min-1. These kinetic correlations support the hypothesis that radical formation results from a reversible reaction between [4Fe-4S]+ and S-adenosylmethionine at the active site to form [4Fe-4S]2+, the 5'-deoxyadenosyl radical, and methionine as intermediates.
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