Lysine 2,3-aminomutase: rapid mix-freeze-quench electron paramagnetic resonance studies establishing the kinetic competence of a substrate-based radical intermediate.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains iron-sulfur clusters and is activated by pyridoxal 5'-phosphate and S-adenosylmethionine, all of which participate in catalysis. Current spectroscopic evidence implicates two substrate-based organic radicals as intermediates in the mechanism. One of these species, the radical N3-(5'-phosphopyridoxylidene)-beta-lysin-2-yl (3), appears in the steady state of the reaction of lysine and has been definitively characterized by EPR and ESEEM spectroscopy. The 2-deuterio form of this radical, 3-2-d, which is generated in the reaction of L-[2-2H]lysine, can be distinguished by line shape analysis from 3. The rate at which the signal for 3-2-d is transformed into that for 3 has been measured by rapid mix-freeze quench kinetic analysis. The rate constant for this process is 24 +/- 8 s-1 at 21 degrees C. This is the rate constant for the turnover of radical 3 and is indistinguishable from the turnover number of lysine 2,3-aminomutase. Therefore, radical 3 is kinetically competent as an intermediate in the reaction of lysine 2,3-aminomutase.