Open AccessSpectroscopic studies on the metal‐ion‐binding sites of Co 2+ ‐substituted D‐xylose isomerase from Streptomyces rubiginosus
Author(s) -
SUDFELDT Christoph,
SCHÄFFER Andreas,
KÄGI Jeremias H. R.,
BOGUMIL Ralf,
SCHULZ HansPeter,
WULFF Stephanie,
WITZEL Herbert
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb19410.x
Subject(s) - chemistry , crystallography , active site , stereochemistry , xylose isomerase , metal , binding site , absorption (acoustics) , octahedron , streptomyces , metal ions in aqueous solution , enzyme , crystal structure , isomerase , materials science , biochemistry , organic chemistry , biology , bacteria , composite material , genetics
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 Co 2+ ‐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 545 , 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 Co 2+ added/mol apoenzyme occupy both sites nearly equally. Subsequently the Co 2+ located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co 2+ /mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd 2+ or Pb 2+ /mol Co 4 ‐loaded derivative displaces the Co 2+ from the B site to form the Pb 4 /Co 4 derivative containing Co 2+ in the A site, reducing activity fourfold while the Pb 4 /Pb 4 species is completely inactive. In contrast, Eu 3+ displaces Co 2+ 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 2+ A‐site spectrum of both the active Co 4 /Co 4 derivative and the less active Pb 4 /Co 4 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.