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首页> 外文会议>12th Symposium on Bioluminescence and Chemiluminescence, Apr 5-9, 2002, Robinson College, University of Cambridge, UK >STRUCTURAL STUDY OF PHOTINUS PYRALIS FIREFLY LUCIFERASE USING FLUORESCENCE
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STRUCTURAL STUDY OF PHOTINUS PYRALIS FIREFLY LUCIFERASE USING FLUORESCENCE

机译：荧光法测定梨状果胶酶的结构研究

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Firefly luciferase (FFL) catalyses the oxidation of a heterocyclic molecule called luciferin (LH_2) in the presence of ATP and oxygen. Reaction proceeds through the formation of a ternary complex followed by an adenylation step in which an intermediate product luciferyl-adenylate is formed. Different observations suggest that FFL undergoes significant conformational changes in the process of the bioluminescent reaction. The crystal structure of FFL without bound substrates was shown to consist of a large N-terminal domain and a small C-terminal domain separated by a wide solvent filled cleft.2 The active centre is considered to be located on the inner surface of the cleft. The conformation of FFL without bound substrates will be further referred to as "opened". All attempts to obtain crystals of FFL with the bound substrates and to resolve its crystal structure by X-ray analysis have failed so far. So there is no direct evidence for the conformational changes. On the other hand it was shown that the crystal structure of phenylalanine-activating subunit of gramicidin synthetase 1, the only FFL homologue with resolved crystal structure, in a complex with phenylalanine, Mg~(2+) and AMP is remarkably similar in size and shape to the FFL "opened" conformation but the C-terminal domain is significantly rotated and approaches the N-terminal domain. It suggests that binding of LH_2 and ATP in the active centre of FFL causes a similar conformational change in its structure which will be further referred to as "closed". Tryptophan (Trp) residue can be used as a sensitive intrinsic fluorescence probe of protein conformational changes, so a catalytically active tryptophan-free mutant (TF) of FFL has been constructed by subsequent site-directed mutagenesis of two naturally occurring Trp residues in positions 417 and 426 (Fig. 1). This has allowed to construct a number of FFL mutants containing single Trp residue in different positions predicted to be sensitive to conformational changes in FFL caused by binding LH_2 and ATP.
机译：萤火虫荧光素酶（FFL）在ATP和氧气存在下催化称为荧光素（LH_2）的杂环分子的氧化。反应通过形成三元络合物进行，随后进行腺苷酸化步骤，其中形成中间产物荧光素-腺苷酸。不同的观察结果表明，FFL在生物发光反应过程中发生了显着的构象变化。没有结合基质的FFL的晶体结构显示为由一个大的N末端结构域和一个小的C末端结构域，该区域由宽的溶剂填充裂隙隔开。2活性中心被认为位于裂隙的内表面。没有结合的底物的FFL的构象将进一步称为“开放的”。迄今为止，所有获得具有结合的底物的FFL晶体并通过X射线分析解析其晶体结构的尝试都失败了。因此，没有直接证据证明构象变化。另一方面，显示了与丙氨酸，Mg〜（2+）和AMP形成的复合物中，短杆菌肽合成酶1的苯丙氨酸激活亚基的晶体结构是唯一的具有分解的晶体结构的FFL同源物。形状为FFL“开放”构象，但C末端结构域显着旋转并接近N末端结构域。这表明LH_2和ATP在FFL活性中心的结合会导致其结构发生类似的构象变化，这将进一步称为“封闭”。色氨酸（Trp）残基可用作蛋白质构象变化的敏感内在荧光探针，因此通过随后在位置417上两个天然存在的Trp残基的定点诱变，构建了FFL的无催化活性的色氨酸突变体（TF）和426（图1）。这已允许构建许多在不同位置含有单个Trp残基的FFL突变体，这些突变体预计对结合LH_2和ATP引起的FFL构象变化敏感。

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