Evidence for Conformational Movement and Radical Mechanism in the Reaction of 4-Thia-l-lysine with Lysine 5,6-Aminomutase

We demonstrate that the steady state reaction of lysine 5,6-aminomutase with substrate analogue 4-thia-l-lysine generates a radical intermediate, which accumulates in the enzyme to an electron paramagnetic resonance (EPR) detectable level. EPR line width narrowing of ∼1 mT due to [4′-²H] labeling of the pyridoxal-5′-phosphate (PLP), an isotropic hyperfine coupling of 40 MHz for the proton at C4′ of PLP derived from ²H electron nuclear double resonance (ENDOR) measurement, and spin density delocalization onto the ³¹P of PLP realized from observations of the ³¹P ENDOR signal provide unequivocal identification of the radical as a substrate−PLP-based species. X- and Q-band EPR spectra fittings demonstrate that this radical is spin coupled with the low spin Co²⁺ in cob(II)alamin and the distance between the two species is about 10 Å. These results provide direct evidence for the active site motion upon substrate binding, bringing the adenosylcobalamin to the proximity of substrate−PLP for subsequent H-atom abstraction and for the notion that lysine 5,6-aminomutase functions by a radical mechanism. Observation of ²H-ENDOR signal also provides a reliable hyperfine coupling constant for future comparison with quantum-mechanical-based calculations to gain further insight into the molecular structure of this steady state radical intermediate.