Lin L N, Brandts J F
Biochemistry. 1983 Feb 1;22(3):559-63. doi: 10.1021/bi00272a006.
Using the method of isomer-specific proteolysis, the isomerization of proline-93 has been monitored directly during the time course of the unfolding and refolding reactions of RNase A. It has been found that proline-93 is 100% cis in the native protein and 70% cis in the reversibly unfolded protein. During the unfolding reaction, the change from 100% to 70% cis occurs as a first-order process with a relaxation time of 140 s in 8.5 M urea, 10 degrees C. For refolding, the change from 70% to 100% cis also occurs as a first-order process, with a relaxation time (10 degrees C) of 90 s in 0.3 M urea, 130 s in 1.0 M urea, and 310 s in 2.0 M urea. Parallel experiments which measured the recovery of enzyme activity during refolding were also conducted. These show that 30% of the activity recovers in a slow phase with a first-order relaxation time (10 degrees C) of 100 s in 0.3 M urea. Because of the excellent agreement of both the amplitude and relaxation time for trans-to-cis isomerization and for activity recovery, it is concluded that the slowest phase in the recovery of enzyme activity is rate limited by the isomerization of proline-93. These results demonstrate that proline-93 must be cis before refolding to the active form can take place, in contrast to previous suggestions, and argue against the existence of a nativelike intermediate form on the refolding pathway which contains proline-93 in the incorrect trans configuration.
采用异构体特异性蛋白水解方法，在核糖核酸酶A展开和重折叠反应的时间进程中，直接监测了脯氨酸-93的异构化过程。结果发现，脯氨酸-93在天然蛋白质中100%为顺式，在可逆展开的蛋白质中70%为顺式。在展开反应过程中，从100%顺式到70%顺式的变化以一级过程发生，在8.5M尿素、10℃条件下的弛豫时间为140秒。对于重折叠，从70%顺式到100%顺式的变化也以一级过程发生，在0.3M尿素中10℃时的弛豫时间为90秒，在1.0M尿素中为130秒，在2.0M尿素中为310秒。还进行了平行实验，测量重折叠过程中酶活性的恢复情况。这些实验表明，30%的活性在一个缓慢阶段恢复，在0.3M尿素中10℃时的一级弛豫时间为100秒。由于反式到顺式异构化以及活性恢复的幅度和弛豫时间都非常吻合，因此得出结论，酶活性恢复中最慢的阶段受脯氨酸-93异构化的速率限制。这些结果表明，与之前的观点相反，脯氨酸-93在重折叠为活性形式之前必须是顺式的，并且反对在重折叠途径中存在一种含有处于错误反式构型的脯氨酸-93的类似天然态的中间形式。
