Topographical characterization of the ubiquinone reduction site of glucose dehydrogenase in Escherichia coli using depth-dependent fluorescent inhibitors

• Published in: Biochimica et biophysica acta Vol. 1412; no. 1; pp. 29 - 36
• Main Authors: Miyoshi, Hideto, Niitome, Yutaka, Matsushita, Kazunobu, Yamada, Mamoru, Iwamura, Hajime
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 26.05.1999
• Subjects: Binding Sites; Capsaicin - chemistry; Capsaicin - pharmacology; Cell Membrane - chemistry; Escherichia coli; Escherichia coli - enzymology; Fluorenes - pharmacology; Fluoresceins; Glucose 1-Dehydrogenase; Glucose dehydrogenase; Glucose Dehydrogenases - antagonists & inhibitors; Glucose Dehydrogenases - chemistry; Oxidation-Reduction; Phosphatidylcholines; Respiratory inhibitor; Ubiquinone; Ubiquinone - chemistry; PC, phosphatidylcholine; Glucose dehydrogenase; DB, 2,3-dimethoxy-5-methyl-6- n-decyl-1,4-benzoquinone; Ubiquinone; FEDA, 1-(2-fluorenyl)ethyl 3,4-dimethoxyphenylacetate; Escherichia coli; θ-DFA, θ-(3,4-dimethoxyphenyl)acetyloxy-7-ethyl-2-fluorenenonanoic acid; GDH, glucose dehydrogenase; α-DFA, α-(3,4-dimethoxyphenyl)acetyloxy-7-nonyl-2-fluoreneacetic acid; PB, 2,3-dimethoxy-5-methyl-6- n-pentyl-1,4-benzoquinone; Q n , ubiquinone-n; Respiratory inhibitor
• ISSN: 0005-2728; 0006-3002; 1879-2650
• DOI: 10.1016/S0005-2728(99)00009-2

Membrane-bound glucose dehydrogenase in Escherichia coli possesses a binding site for ubiquinone as well as glucose, metal ion and pyrroloquinoline quinone. To probe the depth of the ubiquinone binding site in the membrane environment, we synthesized two types of fluorenyl fatty acids which bear an inhibitor mimic moiety (i.e., specific inhibitor capsaicin) close to the fluorene located at different positions in the alkyl tail chain; one close to the polar carbonyl head group (α-(3,4-dimethoxyphenyl)acetyloxy-7-nonyl-2-fluoreneacetic acid, α-DFA), and the other in the middle of the chain (θ-(3,4-dimethoxyphenyl)acetyloxy-7-ethyl-2-fluorenenonanoic acid, θ-DFA). Mixed lipid vesicles consisting of phosphatidylcholine (PC) and α-DFA or θ-DFA were prepared by sonication method, and fluorescent quenching against a hydrophilic quencher, iodide anion, was examined. The vesicles containing α-DFA were more susceptible to quenching than those containing θ-DFA, indicating that the fluorene and consequently capsaicin mimic moiety are located at different depths in the lipid bilayer depending upon the position of attachment to the alkyl tail chain. The purified glucose dehydrogenase was reconstituted into PC vesicles which consisted of PC and α-DFA or θ-DFA with various molar ratios. For both types of reconstituted vesicles, the extent of inhibition of short-chain ubiquinone reduction activity increased with increases in the molar ratio of fluorenyl fatty acid to PC. The ubiquinone reduction activity was more significantly inhibited in the reconstituted vesicles containing α-DFA compared to those containing θ-DFA. Our findings strongly suggested that the ubiquinone reduction site in glucose dehydrogenase is located close to the membrane surface rather than in the hydrophobic membrane interior.