European Journal of Biochemistry, November 1990, Vol.193(3), pp.863-871
The coordination sphere of the two metal‐binding sites/subunit of the homotetrameric D‐xylose isomerase from has been probed by the investigation of the Co‐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, ɛ, of 18 M, 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 ɛ 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 [Callens et al. (1988) , 285–290] having the same feature but lower ɛ values. The first 4 mol Co added/mol apoenzyme occupy both sites nearly equally. Subsequently the Co located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co/mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd or Pb/mol Co‐loaded derivative displaces the Co from the B site to form the Pb/Co derivative containing Co in the A site, reducing activity fourfold while the Pb/Pb species is completely inactive. In contrast, Eu displaces Co preferentially from the A site. Thus, the high‐and low‐affinity sites may be different for different for different cations. After addition of the substrates D‐xylose, D‐glucose and D‐fructose and the inhibitor xylitol the intense Co A‐site spectrum of both the active Co/Co derivative and the less active Pb/Co 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.
Aldose-Ketose Isomerases ; Carbohydrate Epimerases -- Metabolism ; Cobalt -- Pharmacology ; Streptomyces -- Enzymology;