Abstract
Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H+ and Na+ transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a “direct coupling” mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na+ transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K+-dependent H+-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PPi analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PPi hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg2+ (essential cofactor). The replacement of K+, the second metal cofactor, by Na+ increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PPi hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell’s direct coupling with conformational coupling to catalyze cation transport across the membrane.
Highlights
Forms of life appear to have depended on pyrophosphate (PPi ) as the primary energy currency instead of ATP—the so-called “PPi world” [1]
We have earlier demonstrated that the dependence of membrane PPase (mPPase) activity on Mg2 PPi concentration is described by a bell-shaped curve with the maximum at approximately
This observation was explained in terms of the model shown in Scheme 1, assuming the non-identical behavior of two active sites in the homodimeric mPPase
Summary
Forms of life appear to have depended on pyrophosphate (PPi ) as the primary energy currency instead of ATP—the so-called “PPi world” [1]. Contemporary organisms produce PPi from ATP and other nucleoside triphosphates as a byproduct in numerous biosynthetic reactions [2]. All plants and many prokaryotic organisms have retained a relict membrane PPase (mPPase; EC 7.1.3.1, formerly 3.6.1.1), which uses PPi energy to transport H+ and/or Na+ ions across membranes [3,4,5,6,7,8]. This reaction is thermodynamically reversible, and there is controversy about the direction in which mPPase works in cells. An increasing body of evidence indicates that mPPases contribute to the tolerance of plants and the bacteria that live in harsh conditions to abiotic stress, such as salination, drought, cold, anoxia, and nutrient limitation [9,10,11,12]
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