Nau Werner M, Ghale Garima, Hennig Andreas, Bakirci Hüseyin, Bailey David M
School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, D-28759 Bremen, Germany.
J Am Chem Soc. 2009 Aug 19;131(32):11558-70. doi: 10.1021/ja904165c.
A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays ("supramolecular tandem assays") exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products ("substrate-selective assays"). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M(-1) with arginine, 550 M(-1) with ornithine, and 60,000 M(-1) with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 x 10(6) M(-1) with cadaverine, 1.1 x 10(5) M(-1) with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 x 10(5) M(-1) with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a "product-selective" with a "substrate-selective" assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This "domino tandem assay" was performed in the same solution with a single reporter pair (CB7/AO).
适度选择性的主客体结合与酶促转化令人印象深刻的特异性相结合，使得能够在均相溶液中实时监测酶促反应。由此产生的酶分析方法（“超分子串联分析”）利用荧光染料与大环主体的动态结合，与底物和产物的结合相互竞争。研究了两个酶促反应的例子：精氨酸酶催化精氨酸水解为鸟氨酸，以及二胺氧化酶催化尸胺氧化为5-氨基戊醛，其中底物对大环的亲和力高于产物（“底物选择性分析”）。底物的消耗使得荧光染料在酶促反应过程中能够进入大环，从而产生所需的荧光响应。对于精氨酸酶，对磺酸杯[4]芳烃被用作大环，它与精氨酸的结合常数为6400 M⁻¹，与鸟氨酸的结合常数为550 M⁻¹，与所选荧光染料（1-氨甲基-2,3-二氮杂双环[2.2.2]辛-2-烯）的结合常数为60000 M⁻¹；该染料在其络合状态下荧光较弱，这导致在酶促反应过程中荧光响应关闭。对于二胺氧化酶，葫芦[7]脲（CB7）被用作大环，它与尸胺的结合常数为4.5×10⁶ M⁻¹，与1-氨基戊烷（作为热不稳定的1-氨基戊醛的模型）的结合常数为1.1×10⁵ M⁻¹，与所选荧光染料（吖啶橙，AO）的结合常数为2.9×10⁵ M⁻¹；AO在其络合状态下荧光较强，这导致在酶促氧化时荧光响应开启。结果表明，串联分析可成功用于探测酶的抑制作用。估计了添加已知抑制剂后的抑制常数，即精氨酸酶的S-(2-硼乙基)-L-半胱氨酸和2(S)-氨基-6-硼己酸，以及二胺氧化酶的氰化钾。通过将“产物选择性”分析与“底物选择性”分析顺序耦合，还能够监测一个多步生化途径，即赖氨酸脱羧酶将赖氨酸脱羧为尸胺，随后二胺氧化酶将尸胺氧化。这种“多米诺串联分析”是在同一溶液中用一对单一报告分子（CB7/AO）进行的。
