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-β-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 K_m 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 K_m for 4-thialysine is 1.4 ± 0.1 mM, and the maximum velocity V_m = 0.19 ± 0.02 μmol min^-1 mg^-1 at 37°C and pH 8.0. The kinetic parameters for the reaction of lysine under the same conditions are: K_m = 4.2 ± 0.5 mM and V_m = 43 ± 1 μmol min^-1 mg^-1. The discrepancy between K_m and the apparent K_i 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.