Pre‐steady‐state kinetics and solvent isotope effects support the “billiard‐type” transport mechanism in <scp>Na</scp><sup>+</sup>‐translocating pyrophosphatase
Anssi M. Malinen, Viktor A. Anashkin, В. Н. Орлов, Alexander V. Bogachev, Reijo Lahti, Alexander A. Baykov
Abstract
Abstract 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.