Visible, EPR and electron nuclear double-resonance spectroscopic studies on the two metal-binding sites of oxovanadium (IV)-substituted D-xylose isomerase.
The two metal-binding sites of the D-xylose isomerase from Streptomyces rubiginosus were studied using VO2+ as a sensor for the ligand environment. Titration of the tetrameric enzyme with VO2+, followed by EPR spectroscopy and inhibition studies, show that the first four VO2+ equivalents occupy, in analogy to Co2+, Cd2+ and Pb2+, the binding site B. The visible absorption data and the EPR parameters indicate that a nitrogen ligand is involved in the ligand sphere of the high-affinity B site. The low-affinity A site could be studied selectively by blocking the B site with visible and EPR-silent Cd2+. The visible data and EPR parameters for this site are consistent with a ligand environment composed of oxygen donors without nitrogen ligation. The nitrogen coordination in the high-affinity site could be demonstrated by electron nuclear double-resonance (ENDOR) studies of the 4VO2+ enzyme, and was assigned to a histidine ligand. The 14N resonances are interpreted in terms of a quartet with a coupling value of 13.2 MHz. 1H-ENDOR coupling of 1.7 MHz, exchangeable in D2O, has been assigned to the N-H proton of the histidine. Additional proton ENDOR couplings, which are not exchangeable, are due to protons bound to the carbon atoms of the histidine. For the low-affinity binding site, a nitrogen coordination could be definitely excluded by the ENDOR measurements. Exchangeable 1H-ENDOR couplings observed in this sample were assigned to H2O ligands in the vicinity of VO2+. The results closely relate to what is known from X-ray structure. However, the relative affinities for the two binding sites seem not to be the same for different bivalent cations. In mixed metal samples with four VO2+ and four Co2+ equivalents, the VO2+ is distributed between both binding sites. Small changes in the complex geometry of the A site, indicated by different EPR features, seem to occur if the B site is occupied by Co2+ or by Cd2+.
Bogumil
R
R
Institut für Biochemie, Universität Münster, Federal Republic of Germany.
Hüttermann
J
J
Kappl
R
R
Stabler
R
R
Sudfeldt
C
C
Witzel
H
H
D019747:Aldose-Ketose Isomerases; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology; D014638:Vanadates / Q000378:metabolism*
D008024:Ligands; D002104:Cadmium; D003035:Cobalt; D014638:Vanadates; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase