Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.
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Active site mutant, Bisphosphonate, Drug binding, Farnesyl pyrophosphate synthase, Inhibition mechanism, Substrate binding, Bone Density Conservation Agents, Catalysis, Catalytic Domain, Crystallization, Diphosphonates, Drug Evaluation, Preclinical, Geranyltranstransferase, Humans, Hydrogen Bonding, Hydrogen-Ion Concentration, Imidazoles, Inhibitory Concentration 50, Molecular Conformation, Mutation, Nitrogen, Oligonucleotides, Protein Binding, Recombinant Proteins, Threonine, Tyrosine, Zoledronic Acid