Structural aspects of the effectiveness of bisphosphonates as competitive inhibitors of the plant vacuolar proton-pumping pyrophosphatase

The bisphosphonates (general structure PO₃-R-PO₃) competitively inhibit soluble and membrane-bound inorganic pyrophosphatases (PPases) with differing degrees of specificity. Aminomethylenebisphosphonate (AMBP; HC(PO₃)₂NH₂) is a potent, specific inhibitor of the PPase of higher plant vacuoles (V-PPase). To explore the possibility of constructing photoactivatable probes from bisphosphonates to label the active site of V-PPase we analysed the effects of different analogues on the hydrolytic and proton pumping activity of the enzyme. Bisphosphonates with a range of structures inhibited competitively and the effects on PP_i hydrolysis correlated with the effects on proton pumping. Low-molecular-mass bisphosphonates containing hydrophilic groups (α-NH₂ or OH) were the most effective, suggesting that the catalytic site is in a restricted polar pocket. Bisphosphonates containing a benzene ring were less active but the introduction of a nitrogen atom into the ring increased activity. Compounds of the general formula NH₂(CH₂)_nC(PO₃)₂OH were more inhibitory than compounds of the H(CH₂)_nC(PO₃)₂NH₂, NH₂(CH₂)_nC(PO₃)₂NH₂ or OH(CH₂)_nC(PO₃)₂NH₂ series, with activity decreasing as n increased. A nitrogen atom in the carbon chain increased activity but activity was decreased by the presence of an oxygen atom. An analogue with a ring attached via a four-carbon chain, which included an amide linkage and a hydroxy group on the α-carbon atom, inhibited competitively (K_i = 62.0 µM), suggesting that it may be possible to design bisphosphonate inhibitors which contain a photoactivatable azido group for photoaffinity labelling of V-PPase active site.