Membrane-bound proton-translocating pyrophosphatase of Syntrophus gentianae, a syntrophically benzoate-degrading fermenting bacterium.
Syntrophus gentianae is a strictly anaerobic bacterium which ferments benzoate to acetate, CO2 and H2 in the presence of hydrogen-utilizing partner bacteria. Benzoate is activated by a benzoyl CoA ligase enzyme which forms AMP and pyrophosphate as coproducts. Pyrophosphatase activity was found to be largely membrane bound. Pyrophosphate hydrolysis was associated with proton translocation across the cytoplasmic membrane. Proton translocation could be abolished by the protonophor carbonylcyanide p-chlorophenylhydrazone, and could also be coupled to ATP formation in membrane vesicle preparations. The ratio of ATP formation/pyrophosphate hydrolysis was 1:3. The reverse reaction, ATP-dependent pyrophosphate synthesis, was possible with the same coupling stoichiometry. Pyrophosphatase was 90% saturated at 1 mM pyrophosphate; pyrophosphate concentrations higher than 5 mM inhibited enzyme activity. Inhibition studies with ATP and EDTA indicated that MgPPi- was probably the physiological substrate. The optimum temperature was 35 degrees C. In the presence of Mg2+, the enzyme was remarkably heat stable, with 50% of its maximum activity after 10 min at 60 degrees C. Exogenously added pyrophosphate could not be used for energy conservation.
Schöcke
L
L
Fakultät für Biologie, Mikrobielle Okologie, Universität Konstanz, Germany.
Schink
B
B
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001565:Benzoates / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D018920:Coculture Techniques; D011756:Diphosphates / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D019605:Euryarchaeota; D043564:Inorganic Pyrophosphatase; D008274:Magnesium / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001565:Benzoates; D011756:Diphosphates; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D006180:Proton-Translocating ATPases; D008274:Magnesium