N-alkylated 6′-aminoluciferins are bioluminescent substrates for Ultra-Glo and QuantiLum luciferase:: New potential scaffolds for bioluminescent assays
N-alkylated 6′-aminoluciferins are bioluminescent substrates for Ultra-Glo and QuantiLum luciferase:: New potential scaffolds for bioluminescent assays
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DOI：
10.1021/bi800505u
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发表时间：
2008-09-30
期刊：
影响因子：
2.9
通讯作者：
Klaubert, Dieter H.
中科院分区：
文献类型：
--
作者：
Woodroofe, Carolyn C.;Shultz, John W.;Klaubert, Dieter H.
一组6'-烷基化氨基荧光素被证明是Ultra-Glo和QuantiLum荧光素酶的生物发光底物。这些研究表明，工程型和野生型萤火虫荧光素酶在荧光素6'位置耐受的空间体积比以前报道的要大得多。烷基取代基的性质强烈地影响生物发光信号的强度，其强度根据大小、形状和电荷的不同而变化很大。一些化合物被观察到比相应的未取代的6'-氨基荧光素产生更多的光。用Ultra-Glo测定底物的Michaelis-Menten常数表明，这种变化主要是由V-max的差异引起的，相对光单位范围为1.33 × 10(4)至332 × 10(4)，但在某些情况下K-m （0.73-10.8 μ M）也起作用。分子模拟结果表明，侧链与荧光素结合袋底部的氢键网络的相互作用可能影响底物与酶的结合。
A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V-max, ranging from 1.33 x 10(4) to 332 x 10(4) relative light units, but in some cases K-m (0.73-10.8 mu M) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.