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 th e binuclear active center of D-xylose isomerase (XyII) were investigat ed 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+-XyII with the acyclic inhibitor xylitol revealed E PR spectra with well-resolved hyperfine patterns, but with increased z ero 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 obtaine d for the Co2+/Mn2+-XyII and the Cd2+/Mn2+-XyII samples, respectively. These results indicate direct interaction of the xylitol with the Mn2 + in the A-site. More drastic changes are observed with the substrates D-xylose and D-glucose. and with the cyclic inhibitors 5-thio-alpha-D -glucose and 2-desoxy-D-glucose. For Cd2+/Mn2+-XyII, the EPR spectra w ith substrates and cyclic inhibitors are similar to each other but dif ferent from the spectra with the acylic inhibitor xylitol. They exhibi t well-resolved line patterns with a relative large zero field splitti ng, which was estimated to be in the range of D = 65-85 mT in the vari ous complexes. Binding of substrates or of cyclic inhibitors to the Co 2+/Mn2+-XyII yields EPR spectra without resolved hyperfine interaction s, indicative of dipolar interaction between the two paramagnetic meta l ions. This can be explained with a decrease in the metal-metal dista nce. Furthermore, the EPR data strongly suggest that the corresponding metal ion movement is induced by binding of the cyclic conformation o f either substrates or cyclic inhibitors and not by binding of the ext ended form of the sugars.