Farnesyl pyrophosphate synthase: Real-time kinetics and inhibition by nitrogen-containing bisphosphonates in a scintillation assay
Farnesyl pyrophosphate synthase: Real-time kinetics and inhibition by nitrogen-containing bisphosphonates in a scintillation assay
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DOI：
10.1089/adt.2007.057
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发表时间：
2007-04-01
影响因子：
1.8
通讯作者：
Schmid, Andres
中科院分区：
文献类型：
--
作者：
Glickman, J. Fraser;Schmid, Andres
开发了用于酶法尼基焦磷酸（FPP）合酶（FPPS）的混合读取FlashPlate（R）（PerkinElmer，Waltham，MA）测定法，以快速测量由二甲基烯丙基焦磷酸（DMAPP）合成FPP的两个步骤。该测定使用DMAPP或焦磷酸香叶酯（GPP）和[H-3]异戊烯基焦磷酸（[H-3]IPP）作为底物，并通过将产物捕获到磷脂包被的纯化微量滴定板上并在电荷耦合装置成像仪中监测产物形成来测量FPPS催化的这些转化为[H]FPP或[H-3]GPP。Michaelis-Menten参数-k（cat）GPP（38/min）、K-m IPP（0.6 μ M）和K-m GPP（0.7 μ M）-与先前使用困难相分离技术的研究一致。测定了各种含氮双膦酸盐（N-BPs）的50%抑制浓度，也与先前文献一致。在没有先例的情况下，还检测到非N-BP的较弱抑制（5 μ M）。在预孵育研究中，N-BP的效力，特别是唑来膦酸盐，随着时间的推移缓慢增加100倍。通过将GPP与唑来膦酸盐一起加入，可显著逆转该效力偏移。唑来膦酸与IPP相比无竞争力。因此，这些研究与先前的结构和生物活性研究一致，并表明唑来膦酸盐和GPP结合位点之间快速形成低亲和力复合物，随后形成非常紧密的唑来膦酸盐和酶复合物，这排除了GPP的进一步结合。此外，来自催化循环第一步的底物之一DMAPP被鉴定为催化第二步的1 μ M抑制剂，表明FPP两步合成受DMAPP调节。
A mix-and-read FlashPlate (R) (PerkinElmer, Waltham, MA) assay for the enzyme farnesyl pyrophosphate (FPP) synthase (FPPS) was developed to rapidly measure both steps in the synthesis of FPP from dimethylallyl pyrophosphate (DMAPP). The assay used either DMAPP or geranyl pyrophosphate (GPP) and [H-3]isopentenyl pyrophosphate ([H-3]IPP) as substrates, and measured the FPPS-catalyzed conversion of these into [H]FPP or [H-3]GPP by capturing the products onto a phospholipid-coated scintillating microtiter plate and monitoring the product formation in a chargecoupled device imager. The Michaelis-Menten parameters-k(cat) GPP (38/min), K-m IPP (0.6 mu M), and K-m GPP (0.7 mu M)-were consistent with previous studies using difficult phase separation techniques. The 50% inhibitory concentrations of various nitrogen-containing bisphosphonates (N-BPs) were determined and were also consistent with prior literature. Without precedent, weaker inhibition (5,mu M) of the non-N-BPs was also detected. In preincubation studies, the potency of the N-BPs, and specifically zoledronate, increased slowly over time by 100-fold. This potency shift was reversed significantly by the inclusion of GPP with zoledronate. Zoledronate was uncompetitive with respect to IPP. Thus, these studies were consistent with prior structural and thermodynamicthen-nodynami studies, and suggest a rapid formation of a lower-affinity complex between zoledronate and the GPP binding site, followed by the formation of a very tight complex of zoledronate and enzyme, which excludes further binding of GPP. Furthen-nore, one of the substrates from the first step in the catalytic cycle, DMAPP, was identified as a 1 mu M inhibitor of the second step of the catalysis, suggesting that the FPP two-step synthesis is regulated by DMAPP.