ИСТИНА

Membrane-bound inorganic pyrophosphatase (mPPase) resembles F-ATPase in catalyzing H+ and Na+ transport across lipid membranes in plants and prokaryotes but differs both structurally and mechanistically. mPPase is homodimeric, and the transported proton is generated in the active site from the water nucleophile (Mitchell’s “direct coupling”). There are, however, indications that mPPase may additionally use conformational energy, the key in the “indirect” coupling mechanism of F-ATPase. Here we examined kinetically and by molecular dynamics simulations the inhibition of K+-dependent H+-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PPi analogs (imidodiphosphate and C-substituted bisphosphonates). The results demonstrated non-identical behavior of two active sites of dimeric mPPase in reactions with the inhibitors and substrate. Inhibitor binding to one subunit markedly suppressed its binding to the neighboring subunit, increased the Michaelis constant and decreased the catalytic constant for substrate conversion in the neighboring subunit. The nonequivalence of active sites in PPi hydrolysis in terms of the Michaelis but not the catalytic constant vanished at 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. Molecular dynamics simulations defined the structural changes underlying the kinetic and binding cooperativity in mPPase. These findings provide evidence that mPPase combines elements of Mitchell’s direct coupling and Boyer’s conformational coupling mechanisms to catalyze cation transport across membrane