Inhibition of lysine 2,3-aminomutase by the alternative substrate 4-thialysine and characterization of the 4-thialysyl radical intermediate.
Lysine 2,3-aminomutase catalyzes the interconversion of L-lysine and L-beta-lysine. 4-Thia-L-lysine (4-thialysine) is an alternative substrate for Lysine 2,3-aminomutase. The organic free radical that appears in the steady state of the reaction of 4-thialysine is structurally analogous to the first lysine-based radical in the chemical mechanism (Wu, W., Lieder, K. W., Reed, G. H., and Frey, P. A. (1995) Biochemistry 34, 10532-10537). 4-Thialysine is a much more potent inhibitor of the reaction of lysine than would be anticipated on the basis of the value of Km for its reaction as a substrate. 4-Thialysine is here shown to be a competitive reversible inhibitor with respect to L-lysine, displaying an inhibition constant of 0.12 +/- 0.01 mM. The value of Km for 4-thialysine is 1.4 +/- 0.1 mM, and the maximum velocity Vm = 0.19 +/-0.02 micromol min(-1) mg-1 at 37 degrees C and pH 8.0. The kinetic parameters for the reaction of lysine under the same conditions are: Km = 4.2 +/- 0.5 mM and Vm = 43 +/- 1 micromol min(-1) mg(-1). The discrepancy between Km and the apparent Ki for 4-thialysine arises from the fact that the maximal velocity for 4-thialysine is only 0.44% that for L-lysine. The electron paramagnetic resonance spectra of the organic radical generated at the active site from 4-thialysine and those generated from deuterium and 3-13C-labeled forms of 4-thialysine were analyzed by simulation. Based on the resulting hyperfine splitting constants, the conformation and distribution of the unpaired spin of the radical at the active site were evaluated.
Miller
J
J
University of Wisconsin-Madison, 53705, USA.
Bandarian
V
V
Reed
G H
GH
Frey
P A
PA
D001667:Binding, Competitive / Q000187:drug effects; D003013:Clostridium; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D005609:Free Radicals / Q000032:analysis / Q000737:chemistry*; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008968:Molecular Conformation
D005609:Free Radicals; C000204:S-2-aminoethyl cysteine; D019751:Intramolecular Transferases; C021518:lysine 2,3-aminomutase; D008239:Lysine; D003545:Cysteine