Biringer R G, Austin C M, Fink A L
Department of Chemistry, University of California, Santa Cruz 95064.
Biochemistry. 1988 Jan 12;27(1):311-5. doi: 10.1021/bi00401a047.
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.
在pH* 3.0和6.0条件下，于-15℃的35%甲醇冷冻溶剂中，通过催化活性的恢复以及抑制剂结合情况来监测核糖核酸酶A的重折叠动力学。以胞苷2',3'-环一磷酸作为底物测定催化活性；用竞争性抑制剂胞苷2'-单磷酸来确定抑制剂结合情况。在pH* 3.0时，催化活性恢复和抑制剂结合均呈现双相动力学。在pH* 6.0时，催化活性恢复速率为单相，而抑制剂结合速率为双相。对于抑制剂结合和催化活性而言，观察到的速率之一均依赖于pH。在重折叠过程完成时，催化活性完全恢复。这些观察结果可通过慢重折叠核糖核酸酶的两条平行重折叠途径来解释，在次要的慢重折叠途径上存在几种类似天然的、部分折叠的中间状态。特别值得注意的是，存在至少一种这样的物种，它具有抑制剂结合能力但没有催化活性。根据已知的天然结构，这是合理的。此外，推测存在一种中间体，其脯氨酸-93构象错误且只有部分催化活性（为天然活性的42%）。观察到的最慢瞬态归因于该脯氨酸残基的异构化以及完全催化活性的恢复。
