Spectroscopic studies on the metal‐ion‐binding sites of Co²⁺‐substituted D‐xylose isomerase from Streptomyces rubiginosus

Abstract

The coordination sphere of the two metal-binding sites/subunit of the homotetrameric D-XylOSe isomerase from Streptomyces rubiginosus has been probed by the investigation of the Co2+-substituted enzyme using electronic absorption, CD and magnetic circular dichroic spectroscopies in the visible region. The spectrum of the high-affinity site (B site) has an absorption coefficient, E~~~, of 18 M-' cm-', indicating a distorted octahedral complex geometry. The spectrum of the low-affinity site (A site) shows two absorption maxima at 505 nm and 586 nm with E values of 170 M-' cm-' and 240 M-' cm-', respectively, which indicates a distorted tetrahedral or pentacoordinated complex structure as also observed for the enzyme from Streptomyces violaceoruber [Callens et al. (1988) Biochem. J . 250, 285-2901 having the same feature but lower c values. The first 4 mol Co2+ added/ mol apoenzyme occupy both sites nearly equally. Subsequently the Co2+ located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co2+/mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd2+ or Pb2+/mol Co4-loaded derivative displaces the Co2+ from the B site to form the Pb4/Co4 derivative containing Co2+ in the A site, reducing activity fourfold while the Pb4/Pb4 species is completely inactive. In contrast, Eu3+ displaces Co2 ' preferentially from the A site. Thus, the highand low-affinity sites may be different for different cations. After addition of the substrates D-xylose, D-glucose and D-fructose and the inhibitor xylitol the intense Co2+ A-site spectrum of both the active C04/c04 derivative and the less active Pb4/Co4 derivative decreases, indicating that these compounds are bound to the A site, changing the distorted tetrahedral or pentacoordinated symmetry there to a distorted octahedral complex geometry.The intracellular enzyme D-XylOSe isomerase is widely distributed among microorganisms like Streptomyces, Arthrohacter and Bacillus species or other bacteria [l, 21. It catalyzes in vivo the reversible isomerization of u-xylose to D-xylulose in order to utilize D-xylose from xylans as an energy source. In 1957 Marshall and Kooi [3] found that D-xylose isomerase also converts a-D-glucose into a-D-fructose. These stereospecific reactions are of industrial interest for the production of high-fructose corn syrup from starch [4] and for the conversion of hemicellulose to ethanol [5].D-Xylose isomerases from Streptomyces species exist as tetramers consisting of four identical subunits. The evaluation of X-ray crystal structures of Xyl isomerase from S. rubiginosus ATCC 21 175 [6, 71, S. olivochromogenes [8, 91 and S. rubiginosus ATCC 21 132 (previously classified as S. albus) [lo] has shown that the tetramer appears to be a dimer of dimers. This was also found for the Arthrobacter enzyme [l 11. Callens et al. [12] have shown by SDSjPAGE that treatment of the enzyme from S. violaceovuber with 0.1% SDS causes reversible dissociation into active dimers, whereas t...

X‐ and Q‐band EPR studies on the two Mn2+‐substituted metal‐binding sites of d‐xylose isomerase

“…Addition of Zn2+, Cd", or Pb" to the metal-free apoenzyme does not restore activity. However, their addition in the presence of saturating Mg2+ concentrations inhibits the enzyme almost completely [7].…”

“…Addition of Zn2+, Cd", or Pb" to the metal-free apoenzyme does not restore activity. However, their addition in the presence of saturating Mg2+ concentrations inhibits the enzyme almost completely [7].Spectroscopic studies on the Co2+-substituted Xyl isomerase revealed that each subunit contains two different binding sites [7, 121. The high-affinity site of Co2+, denoted as the B site, has a distorted octahedral coordination geometry, whereas the low-affinity site, denoted as the A site, seems to be pentacoordinated.…”

“…Incorporation of Co2+ into the B site does not lead to an active enzyme. Full catalytic activity is reached only after occupation of both binding sites indicating that binding and catalysis need at least a complex formation of substrates and inhibitors with the A-site metal [7,161. It was demonstrated that the B site is also the high-affinity site for Cd2+, Pb2+ and V02+ [7,131, whereas Eu3+ and Sm3+ obviously bind more strongly to the A site [7,14,151.…”

“…Full catalytic activity is reached only after occupation of both binding sites indicating that binding and catalysis need at least a complex formation of substrates and inhibitors with the A-site metal [7, 161. It was demonstrated that the B site is also the high-affinity site for Cd2+, Pb2+ and V02+ [7, 131, whereas Eu3+ and Sm3+ obviously bind more strongly to the A site [7,14, 151. In EPR and electron-nuclear-double-resonance (endor) measurements of the VO'+-substituted Xyl isomerase it has been shown that the B site contains one histidine besides oxygen donors, whereas the coordination sphere of the A site has no nitrogen ligands [13].…”

“…Spectroscopic studies on the Co2+-substituted Xyl isomerase revealed that each subunit contains two different binding sites [7, 121. The high-affinity site of Co2+, denoted as the B site, has a distorted octahedral coordination geometry, whereas the low-affinity site, denoted as the A site, seems to be pentacoordinated.…”