Novel enzymatic activities of the bioluminescent protein, luciferase

荧光素酶是一类在多种陆地和海洋动植物中表达的蛋白质，能够通过生物发光进行信号交流。最为人所熟知的这类系统之一是从美洲萤火虫--石楠身上发现的。荧光素酶的酶活性催化ATP、氧和辅因子荧光素的反应，以可见光子的形式释放能量，以及副产物AMP、焦磷酸和氧化的荧光素。化学发光是探索分子机制最灵敏的方法之一，因为(1)这种酶将ATP的生化能量转化为光的量子产率约为90%，(2)可以使用先进的微光探测系统进行单光子计数，从而能够记录和研究单分子反应中的事件。如上所述，荧光素酶的行为有无法解释的复杂性，特别是，我们注意到当酶暴露于其已知底物ATP时，在其他核苷酸的存在下，如ADP，会发生某些矛盾水平的光产生和非线性。事实上，人们已经推断，这种机制上的不确定性在很大程度上可以通过腺苷酸激酶(又称“肌酸激酶”)对荧光素酶的污染来解释，腺苷酸激酶在提纯荧光素酶的制剂中含量丰富(例如，被萤火虫的飞行肌肉污染，以及从用于产生重组蛋白的细菌的细胞质中污染等)。腺苷酸激酶通过ADP的歧化产生ATP，这将在荧光素酶反应中产生一个不可预见和不受控制的污染光信号，独立于最初存在的ATP，从而混淆了测量的准确性和特异性。 我们试图研究在ADP存在下荧光素酶的催化活性和化学发光机制。我们发现，在某些情况下，ADP的数量超过了ATP，与纯ATP标准的定量预期相比，可能会有显著的过量光输出。为了排除这种明显的假象，我们使用制备柱层析法纯化了几种荧光素酶(从萤火虫提取物和重组材料中)，目的是排除30kD的腺苷酸激酶(腺苷酸激酶分子量约为10kD)。20kD)。在确认了30kD的数量耗尽后，我们发现与ADP相关的荧光素酶光输出伪影仍然存在，这表明腺苷酸激酶污染不是原因。据报道，无机焦磷酸盐(PPI)可以提高荧光素酶的生物发光产量。为了排除ADP在我们的生物发光分析中可能的激活作用是由于所用制剂中存在PPI污染而引起的人工制品，我们用无机焦磷酸酶(PPiase)处理ADP，它能将一个焦磷酸盐分子转化为两个磷酸根离子。PPiase处理虽然改变了光产生的动力学，但对总的光输出没有影响。此外，我们还证明了在ADP中加入外源PPI并没有激活荧光素酶的光产生，而是显著地抑制了荧光素酶的光产生，这种抑制作用可以被PPiase逆转。因此，我们排除了PPI污染产生的伪影。 然后我们假设，荧光素酶本身可能能够仅从ADP获得足够的能量来产生光输出伪像。为了测试这一点，我们开发了一种运行在透明天然凝胶平台上的凝胶内化学发光分析方法。荧光素酶首先是使用透明的天然凝胶从其他潜在的酶污染物中分离出来的。该凝胶随后与纯化的ADP和荧光素孵育，微光成像显示两个离散的条带产生显著的光输出。随后将过量的ATP添加到这种混合物中，表明这些完全相同的带在单独使用ADP时产生的光也在显著增加，而在(过剩的)ATP中也产生了光。在成像过程结束时，该凝胶的免疫印迹证实，发光的条带对荧光素酶呈阳性染色(而对腺苷酸激酶呈阴性染色)，并且对这些条带(来自不用于免疫印迹的凝胶通道)的蛋白质组学分析证实，存在的唯一蛋白质是荧光素酶，没有任何其他蛋白质污染。我们得出结论，荧光素酶本身能够利用ADP的能量产生光输出，而不依赖于预先形成的ATP的存在，可能是通过催化ADP转化为ATP，而后者被用作最终底物。

Luciferases are a class of proteins expressed in a wide variety of terrestrial and marine animal and plant species that enable communication of signals via bioluminescence. One of the most familiar such systems is known from the American firefly, Photinus pyralis. The enzymatic activity of luciferase catalyzes the reaction of ATP, oxygen and the cofactor, luciferin, releasing energy as a visible photon together with the byproducts, AMP, pyrophosphate and oxidized luciferin. Chemiluminescence is one of the most sensitive ways to probe molecular mechanisms because, (1) the conversion of the biochemical energy of ATP into light by this enzyme has a quantum yield of approximately 90%, and (2) single photon counting can be performed using advanced low-light-level detection systems, enabling the recording and study of events from single molecule reactions. As noted above, there are unexplained complexities in the behavior of luciferase, and in particular, we have noted certain paradoxical levels of light production and non-linearity occur when the enzyme is exposed to its known substrate, ATP, in the presence of other nucleotides, such as ADP. Indeed, it has been reasoned that much of this mechanistic uncertainty could be explained by a certain level of luciferase contamination with adenylate kinase (a.k.a., "myokinase"), which is abundant in preparations from which luciferase is purified (e.g., contaminated by the flight muscles in fireflies, as well as from the cytoplasm of bacteria used in recombinant protein generation, etc.). Adenylate kinase produces ATP through the dismutation of ADP, which would produce an unforseen and uncontrolled contaminating light signal in the luciferase reaction independent of the ATP originally present and thus confounding the accuracy and specificity of the measurement. We sought to examine the catalytic activity and chemiluminescence mechanisms of luciferase in the presence of ADP. We found that under certain circumstances where ADP is in quantitative excess over ATP, there can be a significant excess light output vs that expected quantitatively from pure ATP standards. To rule out that adenylate kinase contamination was responsible for this apparent artifact, we purified several luciferases (both from firefly extracts and recombinant material) using preparative column chromatography designed to exclude < 30 kD species (adenylate kinase mw approx. 20 kD). After confirming the quantitative depletion of species < 30 kD, we found that the ADP-related artifact on luciferase light output was still present, indicating that adenylate kinase contamination was not the cause. It has been reported elsewhere that inorganic pyrophosphate (PPi) may increase the output of luciferase bioluminescence. To rule out that the possible activating effect of ADP in our bioluminescence assay was an artifact caused by the presence of PPi contamination in the preparations used, we treated the ADP with inorganic pyrophospatase (PPiase) that converts one molecule of pyrophosphate to two phosphate ions. PPiase treatment although changing the kinetics of the light production had no effect on the total light output. Furthermore, we demonstrated that addition of external PPi to ADP did not activate but instead significantly inhibited luciferase light production and this inhibition was reversed by treatment with PPiase. Thus, we ruled out an artifact produced by PPi contamination. We then hypothesized that luciferase itself might be able to obtain sufficient energy from ADP alone to produce the light-output artifact. In order to test this, we developed an in-gel chemiluminescence assay run on a clear native gel platform. The luciferase was first separated from other potential enzyme contaminants using a clear native gel. This gel was subsequently incubated with purified ADP and luciferin, and low light level imaging showed that 2 discrete bands yielded significant light output. Subsequent addition of an excess of ATP to this mixture showed that these exact same bands that produced light with ADP alone, were also producing substantially increased light with the (excess) ATP. At the end of the imaging procedure, immunoblotting of this gel confirmed that the bands producing light were positively stained for luciferase (and negative for adenylate kinase), and proteomic analysis of these bands (from gel lanes not used for immunoblotting) identified that the only protein present was luciferase without any other protein contamination. We conclude that luciferase itself is capable of using the energy of ADP to produce light output independently of the presence of preformed ATP, possibly by the catalytic conversion of ADP to ATP and the latter being utilized as the ultimate substrate.