title: Observation of a second substrate radical intermediate in the reaction of lysine 2,3-aminomutase: a radical centered on the beta-carbon of the alternative substrate, 4-thia-L-lysine.
abstract: Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of lysine and beta-lysine by a mechanism in which four organic radicals are postulated as intermediates. One of the intermediates has been identified as the alpha-radical of beta-lysine in imine linkage to pyridoxal phosphate (PLP) [Ballinger, M. D., Frey, P. A., & Reed, G. H. (1992) Biochemistry 31, 10782-10788]. We report here the observation of another of the four putative radical intermediates in the reaction of the alternative substrate, 4-thia-L-lysine (S-2-aminoethyl-L-cysteine). 4-Thialysine is a substrate for lysine 2,3-aminomutase. The Km of 4-thialysine is similar to that for lysine, and the Vm is approximately 3% of that for lysine. Upon mixing 4-thialysine with the activated enzyme in the presence of the required cofactor S-adenosylmethionine, followed by freeze-quenching with liquid N2 in the steady state, a strong EPR signal centered at g = 2.003 is observed. This signal exhibits strong hyperfine splitting due to the presence of 13C at carbon-3 of 4-thialysine, and the EPR pattern is narrowed upon the substitution of deuterium at carbon-3. The hyperfine interactions show that the unpaired electron is centered on carbon-3 of 4-thialysine. The hyperfine pattern in the EPR spectrum is also simplified by the use of 4-thia[5,6-2H4]lysine as the substrate, showing either that the spin is partially delocalized through the sulfur intervening between carbons-3 and -5 or that the conformation is such that protons at carbon-6 are close to carbon-3.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705, USA.
authors.forename: W
authors.initials: W
authors.lastname: Wu
authors.forename: K W
authors.initials: KW
authors.lastname: Lieder
authors.forename: G H
authors.initials: GH
authors.lastname: Reed
authors.forename: P A
authors.initials: PA
authors.lastname: Frey
mesh_terms: D000591:Amino Acid Isomerases / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D003903:Deuterium; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D019751:Intramolecular Transferases; D007700:Kinetics; D012436:S-Adenosylmethionine / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: C000204:S-2-aminoethyl cysteine; D012436:S-Adenosylmethionine; D003903:Deuterium; D000591:Amino Acid Isomerases; D019751:Intramolecular Transferases; C021518:lysine 2,3-aminomutase; D003545:Cysteine