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Pre-steady-state kinetics and solvent isotope effects support the “billiard-type” transport mechanism in Na+-translocating pyrophosphataseстатья
Статья опубликована в высокорейтинговом журнале
Статья опубликована в журнале из списка Web of Science и/или Scopus- Авторы: Malinen Anssi M., Anashkin Viktor A., Orlov Victor N., Bogachev Alexander V., Lahti Reijo, Baykov Alexander A.
- Журнал: Protein Science
- Том: 31
- Номер: 9
- Год издания: 2022
- Издательство: Cold Spring Harbor Laboratory Press
- Местоположение издательства: United States
- Первая страница: 1
- Последняя страница: 16
- Номер статьи: e4394
- DOI: 10.1002/pro.4394
- Аннотация: 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 chemicalstep (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 resemblesother 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.Abbreviations:
- Добавил в систему: Байков Александр Андреевич