Quinoprotein glucose dehydrogenase (EC 1.1.99.17) from Acinetobacter calcoaceticus L.M.D. 79.41 was purified to homogeneity. It is a basic protein with an isoelectric point of 9.5 and an Mr of 94,000. Denaturation yields two molecules of PQQ/molecule and a protein with an Mr of 48000, indicating that the enzyme consists of two subunits, which are probably identical because even numbers of aromatic amino acids were found. The oxidized enzyme form has an absorption maximum at 350 nm, and the reduced form, obtained after the addition of glucose, at 338 nm. Since double-reciprocal plots of initial reaction rates with various concentrations of glucose or electron acceptor show parallel lines, and substrate inhibition is observed for glucose as well as for electron acceptor at high concentrations, a ping-pong kinetic behaviour with the two reactants exists. From the plots, Km values for glucose and Wurster's Blue of 22 mM and 0.78 mM respectively, and a Vmax. of 7.730 mumol of glucose oxidized/min per mg of protein were derived. The enzyme shows a broad substrate specificity for aldose sugars. Cationic electron acceptors are active in the assay, anionic acceptors are not. A pH optimum of 9.0 was found with Wurster's Blue and 6.0 with 2,6-dichlorophenol-indophenol. Two types of quinoprotein glucose dehydrogenases seem to exist: type I enzymes are acidic proteins from which PQQ can be removed by dialysis against EDTA-containing buffers (examples are found in Escherichia coli, Klebsiella aerogenes and Pseudomonas sp.); type II enzymes are basic proteins from which PQQ is not removed by dialysis against EDTA-containing buffers (examples are found in A. calcoaceticus and Gluconobacter oxydans).

A soluble cytochrome b was purified from Acinetobacter calcoaceticus L.M.D. 79.41. On the basis of the alpha-band maximum of a reduced preparation, measured at 25 degrees C, it is designated as cytochrome b-562. This cytochrome is a basic monomeric protein (pI 10.2; Mr 18,000), containing one protohaem group per molecule. The reduced form, at 25 degrees C, showed absorption bands at 428, 532 and 562 nm. At 77 K the alpha-band shifted to 560 nm (with a shoulder at 558 nm). The reduced cytochrome did not react with CO. Cytochrome b-562 is most probably (loosely) attached to the outside of the cytoplasmic membrane, since substantial amounts of it, equimolar to quinoprotein glucose dehydrogenase (GDH), were present in the culture medium when cells were grown in the presence of low concentrations of Triton X-100. The midpoint potential at pH 7.0 was found to be +170 mV, a value that was lowered to +145 mV by the presence of GDH. Since the GDH was shown to have a midpoint potential of +50 mV, cytochrome b-562 could function as the natural primary electron acceptor. Arguments to substantiate this view and to propose a role of ubiquinone-9 as electron acceptor for cytochrome b-562 are presented.

SUMMARYQuinoprotein glucose dehydrogenase (GDH; EC 1.1.99.17) was partially purified from cell-free extracts of Acinetobacter culcouceticus LMD79.41. Intact cells of A. culcouceticus LMD79.41 also oxidized these monosaccharides, but not the disaccharides.The difference in substrate specificity can not be explained by impermeability of the outer membrane for disaccharides, since right-side-out membrane vesicles did not oxidize disaccharides either. Destruction of the cytoplasmic membrane strongly affected the catalytic properties of GDH. Not only did the affinity towards some monosaccharides change substantially, but disaccharides also became good substrates upon solubilization of the enzyme. Thus, at least in A. culcouceticus Correspondence to: J.A. Duine, Laboratory of Microbiology and Enzymology,

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When grown on glucose in K+‐limited chemostat culture, or in batch culture with or without 2,4‐dinitrophenol, several strains of Escherichia coli (including the type strain) were found to synthesize a quinoprotein glucose dehydrogenase apoenzyme. The pyridine nucleotides, NAD+ and NADP+, would not serve as cofactor, but activity could be demonstrated upon addition of 2,7,9‐tricarboxy‐1 H‐pyrrolo(2,3‐f)quinoline‐4,5‐dione (PQQ). Thus, in the presence of PQQ, but not in its absence, glucose was oxidized to gluconic acid. A mutant of E. coli PC 1000 was isolated that lacked Enzyme I of the phosphoenolpyruvate phosphotransferase system (PTS) but still synthesized the glucose dehydrogenase apoenzyme. Whereas this mutant would not grow on glucose in the absence of PQQ, it would do so in the presence of low concentrations (1 μM) of this cofactor. On the basis of these observations, it is concluded that the protein (apoenzyme) formed is a genuine glucose dehydrogenase, but that it is not functional in growing cells due to their inability to synthesize the appropriate cofactor (PQQ), at least under these conditions.