Spectroscopic studies on the metal-ion-binding sites of Co2(+)-substituted D-xylose isomerase from Streptomyces rubiginosus.
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, epsilon 545, of 18 M-1 cm-1, 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 epsilon values of 170 M-1 cm-1 and 240 M-1 cm-1, 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, 285-290] having the same feature but lower epsilon 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 high- and 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 Co4/Co4 derivative and the less active Pb4/PCo4 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.
Sudfeldt
C
C
Institute of Biochemistry, University of Münster, Federal Republic of Germany.
Schäffer
A
A
Kägi
J H
JH
Bogumil
R
R
Schulz
H P
HP
Wulff
S
S
Witzel
H
H
D019747:Aldose-Ketose Isomerases; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002413:Cations, Divalent; D002942:Circular Dichroism; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D011487:Protein Conformation; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*
D002413:Cations, Divalent; D046911:Macromolecular Substances; D003035:Cobalt; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase