Lysine 2,3-aminomutase and trans-4,5-dehydrolysine: characterization of an allylic 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 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 (15)N-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-(2)H(2)]lysine or 4,5-dehydro[4,5-(2)H(2)]lysine, and it is dramatically simplified with 4,5-dehydro-[3,3,4,5,6,6-(2)H(6)]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 concentration 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.
Institute for Enzyme Research and Department of Biochemistry, University of Wisconsin-Madison, 53705, USA.
W
W
Wu
S
S
Booker
K W
KW
Lieder
V
V
Bandarian
G H
GH
Reed
P A
PA
Frey
D001665:Binding Sites; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology; D003903:Deuterium / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D005609:Free Radicals / Q000737:chemistry; D006868:Hydrolysis; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008239:Lysine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D008968:Molecular Conformation; D012436:S-Adenosylmethionine / Q000737:chemistry; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005609:Free Radicals; C002194:dehydrolysine; D012436:S-Adenosylmethionine; D003903:Deuterium; D019751:Intramolecular Transferases; C021518:lysine 2,3-aminomutase; D008239:Lysine