CA2-SITES AND ROLES IN SOLUBLE, QUINOPROTEIN (PYRROLOQUINOLINE-QUINONE CONTAINING) GLUCOSE-DEHYDROGENASE( AND ITS SUBSTITUTES HAVE 2 DIFFERENT BINDING)

To investigate the mode of binding and the role of Ca2+ in soluble, py rroloquinoline-quinone (PQQ)-containing glucose dehydrogenase of the b acterium Acinetobacter calcoaceticus (sGDH), the following enzyme spec ies were prepared and their interconversions studied: monomeric apoenz yme (M); monomer with one firmly bound Ca2+ ion (M); dimer consisting of 2 M (D); dimer consisting of 2 M and 2 PQQ (Holo-Y); dimer consis ting of D with 2 PQQ (Holo-X); fully reconstituted enzyme consisting o f Holo-X with two extra Ca2+ ions (Holo) or substitutes for Ca2+ (hybr id Hole-enzymes). D and Hole are very stable enzyme species regarding monomerization and inactivation by chelator, respectively, the bound C a2+ being locked up in such a way that it is not accessible to chelato r. D can be converted into M by heat treatment and the tightly bound Ca2+ can be removed from M with chelator, transforming it into M. Rea ssociation of M to D occurs spontaneously at 20 degrees C; reassociat ion of M to D occurs by adding a stoichiometric amount of Ca2+. Synerg istic effects were exerted by bound Ca2+ and PQQ, each increasing the affinity of the protein for the other component. Dimerization of M to D occurred with Ca2+, Cd2+, Mn2+, and Sr2+ (in decreasing order of eff ectiveness), but not with Mg2+, Ba2+, Co2+, Ni2+ Zn2+, or monovalent c ations. Conversion of inactive Holo-X into active Hole, was achieved w ith Ca2+ or metal ions effective in dimerization. Although it is likel y that activation of Holo-X involves binding of metal ion to PQQ, the spectral and enzymatic activity differences between normal Holo- and h ybrid Hole-enzymes are relatively small. Titration experiments reveale d that the two Ca2+ ions required for activation of Holo-X are even mo re firmly bound than the two required for dimerization of M and anchor ing of PQQ Although the two binding sites related with the dual functi on of Ca2+ show similar metal ion specificity, they are not identical. The presence of two different sites in sGDH appears to be unique beca use in other PQQ-containing dehydrogenases, the PQQ-containing subunit has only one site. Given the broad spectrum of bivalent metal ions ef fective in reconstituting quinoprotein dehydrogenase apoenzymes to act ive holoenzymes, but the limited spectrum for an individual enzyme; th e specificity is not so much determined by PQQ but by the variable met al-ion-binding sites.