OBSERVATION OF A 2ND 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

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 cr-radical of P-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 K-m of 4-thialysine is similar to that for lysine, and the V-m is similar to 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 N-2 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 C-13 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-H-2(4)]lysine as the substrate, showing either that the spin is partially delocalized through the sulfur intervening between carbons-a and -5 or that the conformation is such that protons at carbon-6 are close to carbon-3. The radical appears to be the beta-radical of 4-thialysine, the 4-sulfur analog of the beta-radical of lysine, a precursor to the previously identified alpha-radical of beta-lysine. The observation of the beta-radical derived from 4-thialysine is presumably made possible by the effect of the 4-sulfur in stabilizing the unpaired electron at carbon-3.