Electron paramagnetic resonance of D-xylose isomerase: evidence for metal ion movement induced by binding of cyclic substrates and inhibitors.

The interactions of substrates and inhibitors with the Mn2+ ions in the binuclear active center of d-xylose isomerase (XylI) were investigated by EPR spectroscopy at X- and Q-band frequencies. The metal binding site 1 (A site) was specifically occupied with Mn2+ ions by blocking the high-affinity metal binding site 2 (B-site) either with Co2+ ions, resulting in a catalytically active enzyme, or with Cd2+ or Pb2+ ions yielding an inactive enzyme species. Incubation of both the Co2+/Mn2+- and the Cd2+/Mn2+-XylI with the acyclic inhibitor xylitol revealed EPR spectra with well-resolved hyperfine patterns, but with increased zero field splitting (zfs) parameter D compared to the spectra without inhibitor. D was estimated by spectral simulation of the central −1/2↔1/2 fine structure transition. D values of 33 and 50 mT were obtained for the Co2+/Mn2+-XylI and the Cd2+/Mn2+-XylI samples, respectively. These results indicate direct interaction of the xylitol with the Mn2+ in the A-site. More drastic changes are ...