A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase
A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase
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DOI：
10.1016/j.ab.2009.08.030
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发表时间：
2010-01-01
影响因子：
2.9
通讯作者：
Miziorko, Henry M.
中科院分区：
文献类型：
--
作者：
Skaff, D. Andrew;Miziorko, Henry M.
3-羟基-3-甲基戊二酰辅酶a （HMG-CoA）合成酶催化乙酰辅酶a生物合成类异戊二烯和甾醇的第一个生理不可逆步骤。含有β -内酯的天然产物对酶活性的抑制与甾醇合成的大量减少有关，这表明HMG-CoA合成酶是潜在的药物靶点，并表明鉴定有效的抑制剂将是有价值的。建立了HMG-CoA合成酶的可见波长光谱光度测定方法。该方法使用二硫代双硝基苯甲酸（DTNB）检测辅酶A （CoASH）在酶的乙酰化作用下的释放，该酶在与乙酰乙酰辅酶A缩合形成HMG-CoA产物之前被底物乙酰化。该测定方法利用了重组酶在没有还原剂的情况下的稳定性。它可以缩小到60 μ l的体积，允许使用384孔微孔板，促进化合物文库的高通量筛选。微孔板法测定的酶活性与使用DTNB法（412 nm）进行CoASH生产的常规规模分光光度法（412 nm）或通过监测第二底物乙酰辅酶a （300 nm）的使用所测得的值相当。高通量分析方法已成功用于筛选超过100,000种药物样化合物的文库，并已确定了人类酶的可逆和不可逆抑制剂。(C) 2009爱思唯尔公司版权所有。
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase catalyzes the first physiologically irreversible step in biosynthesis of isoprenoids and sterols from acetyl-CoA. inhibition of enzyme activity by beta-lactone-containing natural products correlates with substantial diminution of sterol synthesis, identifying HMG-CoA synthase as a potential drug target and Suggesting that identification of effective inhibitors would be valuable. A visible wavelength spectro photo metric assay for HMG-CoA synthase has been developed. The assay uses dithiobisnitrobenzoic acid (DTNB) to detect coenzyme A (CoASH) release on acetylation of enzyme by the substrate acetyl-CoA, which precedes condensation with acetoacetyl-CoA to form the HMG-CoA product. The assay method takes advantage of the stability of recombinant enzyme in the absence of a reducing agent. It can be scaled down to a 60 mu l volume to allow the use of 384-well microplates, facilitating high-throughput screening Of Compound libraries. Enzyme activity measured in the microplate assay is comparable to values measured by using conventional scale spectrophotometric assays with the DTNB method (412 nm) for CoASH production or by monitoring the use of a second substrate, acetoacetyl-CoA (300 nm). The high-throughput assay method has been successfully used to screen a library of more than 100,000 drug-like compounds and has identified both reversible and irreversible inhibitors of the human enzyme. (C) 2009 Elsevier Inc. All rights reserved.