Soluble and Membrane-bound Quinoprotein D-Glucose Dehydrogenases of the Acinetobacter calcoaceticus : The Binding Process of PQQ to the Apoenzymes

抄録

Acinetobacter calcoaceticus LMD 79.41 is a unique bacterium containing a soluble quinoprotein D-glucose dehydrogenase (sGDH) in addition to the membrane-bound quinoprotein D-glucose dehydrogenase (mGDH) which is distributed extensively in Gram-negative bacteria. sGDH has been shown to be a distinct enzyme from mGDH, though both enzymes contain a tightly bound pyrroloquinoline quinone (PQQ) as their prosthetic group. In this study, sGDH was detectable in all strains tested of A. calcoaceticus but not in other Gram-negative bacteria tested, indicating that sGDH can be useful as a taxonomic marker for A. calcoaceticus. The binding process of PQQ with both enzymes was examined by using the apoenzymes purified from a PQQ-deficient mutant strain of A. calcoaceticus. sGDH was able to bind two moles of PQQ in one mole of the homodimer with a fairly high affinity. The binding reaction was much faster at alkaline pH than at acidic pH, and required the presence of some divalent cations such as Cd²⁺, Ca²⁺, Sr²⁺, or Mn²⁺. On the other hand, mGDH bound one mol of PQQ in the monomeric enzyme with a relatively slow reacting process, which was optimum at acidic pH and in the presence of different types of divalent cations such as Mg²⁺, Ca²⁺, Zn²⁺, or Sr²⁺ . Thus, it is suggested that sGDH and mGDH have distinct structures around their PQQ binding site. Furthermore, binding of PQQ affects the conformation of both enzymes, which can be shown from the diminishing intrinsic fluorescence of the enzymes and the increase in resistance against proteolysis upon PQQ binding. Data also suggest that the conformational changes caused by PQQ-binding are more dramatic in sGDH than in mGDH. Based on the results obtained, the differences in PQQ-binding mode between the enzymes and the physiological meanings of sGDH are discussed.