Abstract
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.
Highlights
Tsoumpra et al / Bone 81 (2015) 478–486 used drugs for conditions characterized by excessive bone resorption such as Paget's disease [7], multiple myeloma [8], bone metastases [9] and osteoporosis [10,11]
The nitrogen-containing bisphosphonates (N-BP):farnesyl pyrophosphate synthase (FPPS) interactions are complex in nature due to N-BPs having two different modes of inhibition: a rapid and reversible competitive inhibition with regard to the allylic substrate dimethylallyl pyrophosphate (DMAPP)/GPP, reflecting the inhibition constant Ki, and an uncompetitive or mixed-type inhibition in relation to isopentenyl pyrophosphate (IPP) [16,19]
The latter is a time-dependent inhibition, arising due to an enzyme isomerization occurring in two stages: i) N-BP binds to the allylic site and forms the IPP binding site and ii) IPP binds to the FPPS:N-BP complex and closes the active site using amino acid residues found in the C-terminus of FPPS (Lys350, Arg351, Arg352, Lys353) [16,17]
Summary
The N-BP:FPPS interactions are complex in nature due to N-BPs having two different modes of inhibition: a rapid and reversible competitive inhibition with regard to the allylic substrate DMAPP/GPP, reflecting the inhibition constant Ki, and an uncompetitive or mixed-type inhibition in relation to IPP [16,19] The latter is a time-dependent inhibition, arising due to an enzyme isomerization occurring in two stages: i) N-BP binds to the allylic site and forms the IPP binding site and ii) IPP binds to the FPPS:N-BP complex and closes the active site using amino acid residues found in the C-terminus of FPPS (Lys350, Arg351, Arg352, Lys353) [16,17]. This has been enabled by the generation of the threonine-to-serine (T201S) and tyrosine-to-phenylalanine (Y204F) mutants, which we hypothesised would not disrupt the proposed Thr201:N-BP hydrogen bond and hydrophobic interactions respectively whereas a Thr201 to alanine (T201A) and Tyr204 to alanine (Y204A) mutant should abolish such interactions
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