Tsoumpra Maria K, Muniz Joao R, Barnett Bobby L, Kwaasi Aaron A, Pilka Ewa S, Kavanagh Kathryn L, Evdokimov Artem, Walter Richard L, Von Delft Frank, Ebetino Frank H, Oppermann Udo, Russell R Graham G, Dunford James E
Botnar Research Centre, Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, UK; Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
Bone. 2015 Dec;81:478-486. doi: 10.1016/j.bone.2015.08.020. Epub 2015 Aug 28.
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.
法尼基焦磷酸合酶（FPPS）是含氮双膦酸盐（N-BPs）的主要分子靶点，N-BPs在临床上用作骨吸收抑制剂。我们通过对突变的FPPS蛋白进行动力学和晶体学研究，调查了苏氨酸201（Thr201）和酪氨酸204（Tyr204）残基在底物结合、催化以及N-BPs抑制作用中的作用。Thr201突变体表明甲基在辅助异戊烯基焦磷酸（IPP）结合位点形成中的重要性，而Tyr204突变揭示了该残基在催化和IPP结合中的未知作用。Thr201与N-BP侧链氮之间的相互作用对于唑来膦酸（ZOL）和利塞膦酸（RIS）的紧密结合抑制很重要，尽管RIS仍能与Lys200的主链羰基相互作用。RIS与Tyr204苯环的相互作用被证明对于维持异构化的酶-抑制剂复合物至关重要。对RIS构象受限类似物的研究再次证实了Thr201在与N-BPs形成氢键中的重要性。总之，我们确定了N-BPs抑制FPPS的新特征，并揭示了活性位点残基在催化和底物结合中的未知作用。
