Dataset Information

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 [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 H₂ O with D₂ 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.

SUBMITTER: Malinen AM

PROVIDER: S-EPMC9405524 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Pre-steady-state kinetics and solvent isotope effects support the "billiard-type" transport mechanism in Na+ -translocating pyrophosphatase.

Malinen Anssi M AM Anashkin Viktor A VA Orlov Victor N VN Bogachev Alexander V AV Lahti Reijo R Baykov Alexander A AA

Protein science : a publication of the Protein Society 20220901 9

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 ...[more]

PMID: 36040263

Similar Datasets

Project description:Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ?-75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ?+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.

Dataset Information

Pre-steady-state kinetics and solvent isotope effects support the "billiard-type" transport mechanism in Na⁺ -translocating pyrophosphatase.

Publications

Pre-steady-state kinetics and solvent isotope effects support the "billiard-type" transport mechanism in Na<sup>+</sup> -translocating pyrophosphatase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets