KINETICS OF NUCLEOTIDE-INDUCED CHANGES IN THE TRYPTOPHAN FLUORESCENCE OF THE MOLECULAR CHAPERONE HSC70 AND ITS SUBFRAGMENTS SUGGEST THE ATP-INDUCED CONFORMATIONAL CHANGE FOLLOWS INITIAL ATP BINDING
KINETICS OF NUCLEOTIDE-INDUCED CHANGES IN THE TRYPTOPHAN FLUORESCENCE OF THE MOLECULAR CHAPERONE HSC70 AND ITS SUBFRAGMENTS SUGGEST THE ATP-INDUCED CONFORMATIONAL CHANGE FOLLOWS INITIAL ATP BINDING
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DOI：
10.1021/bi00036a040
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发表时间：
1995-09-12
期刊：
影响因子：
2.9
通讯作者：
MCKAY, DB
中科院分区：
文献类型：
--
作者：
HA, JH;MCKAY, DB
测定了重组牛70 kDa热休克同源蛋白（Hsc 70）的核苷酸诱导色氨酸荧光变化的动力学，Hsc 70是一个60 kDa的亚片段（氨基酸残基1-554），具有ATP酶和肽结合活性，而44 kDa的亚片段（氨基酸残基1-386）仅具有ATP酶活性。由ATP结合Hsc 70和60 kDa片段引起的荧光变化是双相的，并且可以被解释为由两步过程引起的，其中ATP最初在双分子反应中结合，随后是蛋白质-MgATP复合物的构象变化。ADP结合导致的荧光变化表明一个单步，双分子过程。在ATP酶反应的单循环条件下，观察到荧光变化，其速率常数与Hsc 70中的产物释放相关，并且与60 kDa片段中的产物释放/ATP水解（其在单循环条件下在动力学上不可区分）相关。这些数据支持Hsc 70的一个方案，其中初始ATP结合后，但在水解前诱导的构象转变，并通过产品释放诱导的反向转换。60 kDa片段显示出与Hsc 70定量相似的行为。44 kDa ATP酶片段不显示ATP结合的双相动力学，也不显示提示Hsc 70和60 kDa片段中所见的构象变化类型的荧光变化。
The kinetics of nucleotide-induced changes of tryptophan fluorescence have been measured for recombinant bovine 70 kDa heat shock cognate protein (Hsc70), a 60 kDa subfragment (amino acid residues 1-554) which has ATPase and peptide binding activities, and a 44 kDa subfragment (residues 1-386) which has only ATPase activity. The fluorescence changes resulting from ATP binding to Hsc70 and the 60 kDa fragment are biphasic, and can be interpreted as arising from a two-step process in which ATP initially binds in a bimolecular reaction, followed by a conformational change of the protein-MgATP complex. Fluorescence changes resulting from ADP binding indicate a single-step, bimolecular process. Under single-cycle conditions of the ATPase reaction, a fluorescence change is observed whose rate constant correlates with product release in Hsc70, and with product release/ATP hydrolysis (which are kinetically indistinguishable under single-cycle conditions) in the 60 kDa fragment. These data support a scheme for Hsc70 in which a conformational transition is induced after initial ATP binding but prior to hydrolysis, and the reverse transition is induced by product release. The 60 kDa fragment shows behavior that is quantitatively similar to that of Hsc70. The 44 kDa ATPase fragment does not show biphasic kinetics for ATP binding, and does not show fluorescence changes that suggest conformational changes of the type seen in Hsc70 and the 60 kDa fragment.