Intermediates in the refolding of ribonuclease at subzero temperatures. 2. Monitoring by inhibitor binding and catalytic activity.
Intermediates in the refolding of ribonuclease at subzero temperatures. 2. Monitoring by inhibitor binding and catalytic activity.
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零下温度下核糖核酸酶重折叠的中间体。
DOI：
10.1021/bi00401a047
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
A. Fink
中科院分区：
文献类型：
--
作者：
R. Biringer;C. M. Austin;A. Fink
核糖核酸酶A的重折叠动力学通过在-15 ° C下在pH* 3.0和6.0的35%甲醇低温溶剂中催化活性和抑制剂结合的恢复来监测。用胞苷2 '，3'-环状单磷酸作为底物测量催化活性;用竞争性抑制剂胞苷2 '-单磷酸测定抑制剂结合。在pH* 3.0下观察到催化活性和抑制剂结合的恢复的双相动力学。在pH* 6.0的催化活性的回报率是单相的，而抑制剂结合是双相的。对于抑制剂结合和催化活性，所观察到的速率之一是pH依赖性的。在重折叠过程完成时获得催化活性的完全恢复。的观察被解释在两个平行的路径的重折叠慢重折叠核糖核酸酶，与几个本地样，部分折叠的中间状态的次要慢重折叠路径。特别值得注意的是存在至少一种这样的物质，其具有结合能力但不具有催化活性。这可以根据已知的天然结构来合理化。此外，假设中间体具有不正确的Pro-93构象和仅部分催化活性（天然的42%）。观察到的最慢的瞬态归因于该脯氨酸残基的异构化和完全催化活性的恢复。
The kinetics of refolding of ribonuclease A were monitored by the return of catalytic activity and inhibitor binding at -15 degrees C in 35% methanol cryosolvent at pH* 3.0 and 6.0. Catalytic activity was measured with cytidine 2',3'-cyclic monophosphate as substrate; inhibitor binding was determined with the competitive inhibitor cytidine 2'-monophosphate. Biphasic kinetics were observed at pH* 3.0 for both return of catalytic activity and inhibitor binding. At pH* 6.0 the rate of return of catalytic activity was monophasic, whereas that of inhibitor binding was biphasic. For both inhibitor binding and catalytic activity one of the observed rates was pH-dependent. Full return of catalytic activity was obtained at the completion of the refolding process. The observations are interpreted in terms of two parallel pathways of refolding for slow-refolding ribonuclease, with several native-like, partially folded intermediate states on the minor slow-refolding pathway. Of particular note is the presence of at least one such species that has inhibitor-binding capacity but not catalytic activity. This may be rationalized in terms of the known native structure. In addition, an intermediate is postulated which has the incorrect Pro-93 conformation and only partial catalytic activity (42% of the native). The slowest observed transient is attributed to the isomerization of this proline residue and return of full catalytic activity.
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使用质子核磁共振观察核糖核酸酶 A 低温折叠过程中的中间体。
影响因子：
2.9
作者：
Biringer,RG;Fink,AL
通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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核糖核酸酶 A 热解折叠中的甲醇稳定中间体。通过 1H 核磁共振表征。
影响因子：
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作者：
Biringer,RG;Fink,AL
通讯作者：
Fink,AL
核糖核酸酶 A 解折叠和重折叠过程中脯氨酸 93 的异构化。
影响因子：
2.9
作者：
Lin,LN;Brandts,JF
通讯作者：
Brandts,JF