Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase.
Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase.
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发表时间：
1994-04
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通讯作者：
Joachim Weber;S. Wilke-Mounts;Ernst Grell;A. E. Senior
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作者：
Joachim Weber;S. Wilke-Mounts;Ernst Grell;A. E. Senior
以色氨酸荧光为工具研究了大肠杆菌F1-ATPase的非催化核苷酸结合部位。定点突变、亲和标记和LIN-苯并-ATP结合研究表明，αR365和βY354残基位于结合核苷酸的碱基附近；在这里，我们将每个残基突变为色氨酸。新的色氨酸发出荧光信号，表明在空置地点处于高极性(阿尔法W365)或中极性(贝塔W354)的环境中。通过与ATP或ADP的结合，Alpha W365荧光被完全猝灭，提供了一个直接的、特异的非催化位点核苷酸占有率的探针。利用这个信号，我们测量了ATP和ADP的结合参数，表明核苷酸结合是镁依赖的，GTP和ITP确实有一定程度的结合，而AMP、GDP和IDP不结合。在相同的条件下，镁ATP水解酶和非催化位结合的初始速率是可能的；结果表明，非催化位不是催化所必需的。βW354的荧光可被联苯并三磷酸腺苷完全猝灭，但仅被三磷酸腺苷或二磷酸腺苷轻微猝灭。可能，残基β354不像残基α365那样与结合的ATP和ADP的腺嘌呤环紧密并列。以α-W365或β-W354为供体，以催化位点结合的LIN-苯并-ADP为受体，未检测到荧光共振能量转移，表明非催化中心与催化中心之间的距离为>；或=27A。
Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites. alpha W365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy. Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis. Fluorescence from beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365. With either alpha W365 or beta W354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non-catalytic and catalytic sites is > or = 27 A.