Membrane-bound proton-translocating pyrophosphatase of Syntrophus gentianae, a syntrophically benzoate-degrading fermenting bacterium

Abstract

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 pchlorophenylhydrazone, 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 MgPP-i was probably the physiological substrate. The optimum temperature was 35°C. In the presence of Mg2+, the enzyme was remarkably heat stable, with 50% of its maximum activity after 10 min at 60°C. Exogenously added pyrophosphate could not be used for energy conservation.