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

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 [32P]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 H2O with D2O 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.

The Mechanism of Energy Coupling in H+/Na+-Pumping Membrane Pyrophosphatase—Possibilities and Probabilities

“…Alternatively, the EPP → EPP* conversion may be rate-limiting and all subsequent steps, including bound PP i breakdown, be much faster. Both alternatives are consistent with the linear pre-steady-state kinetics, but the mechanism with the rate-limiting chemical step is also favored by the high kinetic D/H isotope effect determined by comparing hydrolysis rates in D 2 O and H 2 O [ 23 ].…”

“…Accordingly, the two P i s leave the active site as mono-magnesium complexes. Recent pulse-chase measurements of PP i binding to Tm-mPPase have revealed that the reaction sequence additionally involves enzyme isomerization before the hydrolysis step [ 23 ], as in soluble pyrophosphatase [ 24 ]. A corresponding extended variant of the reaction sequence is shown in Scheme 1 B.…”

“…Rapid kinetics of PP i hydrolysis was measured only for T. maritima Na + -PPase [ 23 ]. Quenched-flow measurements of P i formation during the first 300 ms of the hydrolysis reaction, corresponding to approximately three turnovers, after a rapid (<5 ms) mixing of mPPase with a large excess of PP i in the presence of Na + or Na + and K + yielded linear product versus time dependencies [ 23 ] ( Figure 2 A). No PP i hydrolysis was observed in the absence of Na + , irrespective of whether K + was present or absent.…”

“…A product burst would indicate a significant contribution of the product release steps, whereas a lag would indicate the contribution of a preceding conformational change. If the reaction involves a conformational change in the enzyme–substrate complex ( Scheme 1 B) [ 23 ], the hydrolysis step may be similarly rate-limiting and all other steps, including the conformational change, very fast. Alternatively, the EPP → EPP* conversion may be rate-limiting and all subsequent steps, including bound PP i breakdown, be much faster.…”

“…Hence, the transported cation (Na + in Tm-mPPase) should bind before or during the rate-limiting step but not after it. Pulse-chase measurements indicated no Na + requirement for PP i binding by Tm-mPPase [ 23 ]. In these experiments, the enzyme was rapidly mixed with a small amount of 32 PP i in the absence of Na + to disallow PP i hydrolysis, and the 32 PP i binding reaction was allowed to proceed for 5–100 ms before it was arrested by adding an excess of nonlabelled PP i (450 µM final concentration) and NaCl.…”