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, ɛ545, of 18 M−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 ɛ 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 ɛ 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 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/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. Copyright © 1990, Wiley Blackwell. All rights reserved