TOPOLOGICAL ANALYSIS OF QUINOPROTEIN GLUCOSE-DEHYDROGENASE IN ESCHERICHIA-COLI AND ITS UBIQUINONE-BINDING SITE

Topological structure of quinoprotein glucose dehydrogenase in the inn er membrane of Escherichia coli was determined by constructing protein fusions with alkaline phosphatase or beta-galactosidase. Analysis of the fusions revealed that the dehydrogenase possesses five membrane-sp anning segments, and the N-terminal and C-terminal portions resided at the cytoplasmic and periplasmic side of the membrane, respectively. T hese results agreed with the hydropathy profile based on its primary s tructure. The topological structure suggests that the predicted bindin g site of the prosthetic group pyrroloquinoline quinone is located at the periplasmic side and that the amino acid residues corresponding to those that were presumed to interact with ubiquinone in one subunit o f mitochondrial NADH dehydrogenase also occur at the periplasmic side. When the purified glucose dehydrogenase and cytochrome o ubiquinol ox idase were reconstituted together with ubiquinone into liposomes, a me mbrane potential could be generated by the electron transfer at the si te of the ubiquinol oxidase but not of the dehydrogenase. These result s suggest that glucose dehydrogenase has a ubiquinone reacting site cl ose to the periplasmic side of the membrane, and thus its electron tra nsfer to ubiquinone appears to be incapable of forming a proton electr ochemical gradient across the inner membrane of E. coli.