Inhibition of lysine 2,3-aminomutase by the alternative substrate 4-thialysine and characterization of the 4-thialysyl radical intermediate

J. Miller, Vahe Bandarian, G. H. Reed, P. A. Frey

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

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 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 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: Km = 4.2 ± 0.5 mM and Vm = 43 ± 1 μmol 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.

Original languageEnglish (US)
Pages (from-to)281-288
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume387
Issue number2
DOIs
StatePublished - Mar 15 2001
Externally publishedYes

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lysine 2,3-aminomutase
Lysine
Substrates
Catalytic Domain
S-2-aminoethyl cysteine
Biochemistry
Deuterium
Electron Spin Resonance Spectroscopy

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Inhibition of lysine 2,3-aminomutase by the alternative substrate 4-thialysine and characterization of the 4-thialysyl radical intermediate. / Miller, J.; Bandarian, Vahe; Reed, G. H.; Frey, P. A.

In: Archives of Biochemistry and Biophysics, Vol. 387, No. 2, 15.03.2001, p. 281-288.

Research output: Contribution to journalArticle

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abstract = "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 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 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: Km = 4.2 ± 0.5 mM and Vm = 43 ± 1 μmol 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.",
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