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 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+-XylI, the EPR spectra with substrates and cyclic inhibitors are similar to each other but different from the spectra with the acylic inhibitor xylitol. They exhibit well-resolved line patterns with a relative large zero field splitting, which was estimated to be in the range of D = 65-85 mT in the various complexes. Binding of substrates or of cyclic inhibitors to the Co2+/ Mn2+-XylI yields EPR spectra without resolved hyperfine interactions, indicative of dipolar interaction between the two paramagnetic metal ions. This can be explained with a decrease in the metal-metal distance. Furthermore, the EPR data strongly suggest that the corresponding metal ion movement is induced by binding of the cyclic conformation of either substrates or cyclic inhibitors and not by binding of the extended form of the sugars.
Bogumil
R
R
Institut für Biochemie, Universität Münster, Germany.
Kappl
R
R
Hüttermann
J
J
Witzel
H
H
D019747:Aldose-Ketose Isomerases; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent; D003035:Cobalt / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014993:Xylitol / Q000494:pharmacology; D014994:Xylose / Q000378:metabolism
D002413:Cations, Divalent; D008670:Metals; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D014994:Xylose; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D014993:Xylitol