Flash properties of Gaussia luciferase are the result of covalent inhibition after a limited number of cycles
Flash properties of Gaussia luciferase are the result of covalent inhibition after a limited number of cycles
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DOI：
10.1002/pro.4023
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发表时间：
2021-01-23
期刊：
影响因子：
8
通讯作者：
Winther, Jakob R.
中科院分区：
文献类型：
--
作者：
Dijkema, Fenne Marjolein;Nordentoft, Matilde Knapkoien;Winther, Jakob R.
荧光素酶被广泛用作基因表达和敏感检测系统的报告者。来自海洋桡足动物Gaussia princeps的荧光素酶（GLuc）已经得到了普及，主要是因为它是分泌的，并显示出非常高的光强度。萤火虫荧光素酶的动力学行为与传统的稳态Michaelis-Menten动力学一致，而GLuc表现出所谓的“闪光”动力学，这意味着光发射的爆发，随后是快速衰减。由于这种行为的机制背景尚不清楚，我们决定更详细地破译它。我们表明，光信号的衰减不是由于底物的消耗，而是由酶的不可逆失活引起的。失活发生在10到200个反应循环后，取决于底物浓度，可以用两个指数和相关的速率常数来描述。其中，主导因子随底物浓度线性增加，次要因子与底物浓度无关。在初始发光反应速率方面，随着底物浓度的增加而增加到1.5倍，并且在10 μ M coelenterazine时没有饱和迹象。最后，我们发现酶的失活形式在尺寸排除层析和SDS-PAGE分析中具有更大的表观尺寸，并且在333 nm激发时在410 nm处出现荧光峰。这些发现表明，在催化过程中，高斯荧光素酶的“闪光”动力学是由与底物衍生物的不可逆共价结合引起的。
Luciferases are widely used as reporters for gene expression and for sensitive detection systems. The luciferase (GLuc) from the marine copepod Gaussia princeps, has gained popularity, primarily because it is secreted and displays a very high light intensity. While firefly luciferase is characterized by kinetic behavior which is consistent with conventional steady-state Michaelis-Menten kinetics, GLuc displays what has been termed "flash" kinetics, which signify a burst in light emission followed by a rapid decay. As the mechanistic background for this behavior was unclear, we decided to decipher this in more detail. We show that decay in light signal is not due to depletion of substrate, but rather is caused by the irreversible inactivation of the enzyme. Inactivation takes place after between 10 and 200 reaction cycles, depending on substrate concentration and can be described by the sum of two exponentials with associated rate constants. The dominant of these increases linearly with substrate concentration while the minor is substrate-concentration independent. In terms of rate of initial luminescence reaction, this increases with the substrate concentration to the power of 1.5 and shows no signs of saturation up to 10 mu M coelenterazine. Finally, we find that the inactivated form of the enzyme has a larger apparent size in both size exclusion chromatography and SDS-PAGE analysis and shows a fluorescence peak at 410 nm when excited at 333 nm. These findings indicate that the "flash" kinetics in Gaussia luciferase are caused by an irreversible covalent binding to a substrate derivative during catalysis.