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, CO, and H-2 in the presence of hydrogen-utilizing partner bacteria. Benzoate is activated by a benzoyl CoA ligase enzym e which forms AMP and pyrophosphate as coproducts, Pyrophosphatase act ivity was found to be largely membrane bound. Pyrophosphate hydrolysis was associated with proton translocation across the cytoplasmic membr ane. Proton translocation could be abolished by the protonophor carbon ylcyanide p-chlorophenylhydrazone, and could also be coupled to ATP fo rmation in membrane vesicle preparations. The ratio of ATP formation/p yrophosphate hydrolysis was 1:3. The reverse reaction, ATP-dependent p yrophosphate synthesis, was possible with the same coupling stoichiome try. Pyrophosphatase was 90% saturated at 1 mM pyrophosphate; pyrophos phate concentrations higher than 5 mM inhibited enzyme activity. Inhib ition 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 add ed pyrophosphate could not be used for energy conservation.