X- and Q-band EPR studies on the two Mn(2+)-substituted metal-binding sites of D-xylose isomerase.
The two metal-binding sites (A and B)/subunit of the homotetrameric D-xylose isomerase (Xyl isomerase) from Streptomyces rubiginosus have been studied with Mn(2+)-EPR spectroscopy at X-band and Q-band frequencies and with electronic spectroscopy. Displacement studies in the visible absorbance range showed that Mn2+ have a higher affinity for the B site. With the low-affinity A site unoccupied, the coordination sphere of Mn2+ in the B site is quite distorted giving rise to a highly anisotropic X-band EPR spectrum. Simulation of the Q-band spectrum reveals a zero field splitting (zfs) D of about 45-48 mT and a rhombicity parameter E/D between 0.2 and 0.3. Occupation of both binding sites with Mn2+ induces a significant shift towards a higher symmetry in the coordination sphere of the B site resulting in similar zfs parameters for both binding sites. The change in A-site environment caused by B-site occupation was analysed in mixed Xyl isomerase derivatives, in which the B site is loaded with Co2+, Cd2+ or Pb2+ and the A site with Mn2+. In the Co2+/Mn2+ Xyl isomerase the Mn2+ has a relatively symmetric ligand environment with small zfs parameters (D = 12 mT, E/D < 0.15). Substituting Co2+ with Cd2+ or Pb2+ in the B site leads to a drastic increase in the zfs parameters of Mn2+ in the A site. The distortions are directly linked to the ionic radii of the ions bound to the B site and may be mediated by the carboxylate group of Glu216 that bridges the metal-binding sites. The EPR spectra also reflect the catalytic activity of the mixed metal samples. With the larger Cd2+ or Pb2+ in the B site, which are strongly influencing the stereochemistry of the A site, the catalytic activity is lost, whereas Co2+ and Mn2+ render the enzyme in an active state, so that the mutual influence on catalysis depends on the complex geometry of both metal-binding sites.
Institut für Biochemie, Universität Münster, Germany.
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Bogumil
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Kappl
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Hüttermann
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Sudfeldt
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Witzel
D019747:Aldose-Ketose Isomerases; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007854:Lead / Q000378:metabolism; D008345:Manganese / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology
D002104:Cadmium; D007854:Lead; D003035:Cobalt; D008345:Manganese; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase