title: Structural aspects of the effectiveness of bisphosphonates as competitive inhibitors of the plant vacuolar proton-pumping pyrophosphatase.
abstract: 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 PPi hydrolysis correlated with the effects on proton pumping. Low-molecular-mass bisphosphonates containing hydrophilic groups (alpha-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 alpha-carbon atom, inhibited competitively (Ki=62.0 microM), suggesting that it may be possible to design bisphosphonate inhibitors which contain a photoactivatable azido group for photoaffinity labelling of V-PPase active site.
authors.affiliation: Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, U.K. ruth.gordon-weeks@bbsrc.ac.uk
authors.forename: R
authors.initials: R
authors.lastname: Gordon-Weeks
authors.forename: S
authors.initials: S
authors.lastname: Parmar
authors.forename: T G
authors.initials: TG
authors.lastname: Davies
authors.forename: R A
authors.initials: RA
authors.lastname: Leigh
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004164:Diphosphonates / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007887:Fabaceae / Q000201:enzymology*; D043564:Inorganic Pyrophosphatase; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010946:Plants, Medicinal; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004164:Diphosphonates; D004791:Enzyme Inhibitors; D010940:Plant Proteins; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase