Pre‐steady‐state kinetics and solvent isotope effects support the “billiard‐type” transport mechanism in Na (+)‐translocating pyrophosphatase

Membrane‐bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H(+) pump that transports the H(+) ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na(+) via a hypothetical “billiard‐type” mechanism, also involving the hydrolysis‐generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid‐quench and pulse‐chase measurements with [(32)P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate‐limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme‐substrate complex. Na(+), whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H(2)O with D(2)O decreased the rates of pyrophosphate hydrolysis by both Na(+)‐ and H(+)‐transporting bacterial mPPases, the effect being more significant than with a non‐transporting soluble pyrophosphatase. We also show that the Na(+)‐pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis‐generated proton both in H(+)‐pumping and Na(+)‐pumping by mPPases.