Choi Kee-Hyun, Licht Stuart
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Biochemistry. 2005 Oct 25;44(42):13921-31. doi: 10.1021/bi0505060.
Processive proteases can unfold proteins and cleave them into fragments of a characteristic size. The detailed mechanism by which product sizes are controlled is still in question. One possible mechanism for the control of product sizes would be translocation of unfolded polypeptides to the protease active sites in units of defined length. We have investigated the mechanism by which ClpAP, an energy-dependent protease from Escherichia coli, controls the sizes of its peptide products. We show that ClpAP generates peptide products with a distribution of sizes that has a pronounced peak at a peptide length of 6-8 amino acid residues. This product size distribution, which is similar to that observed previously for the proteasome, is robust to perturbations that interfere with translocation or proteolysis. To explain these results, we propose a mechanism in which translocation alternates with proteolysis, allowing peptides of more or less uniform length to be cleaved processively from a translocating substrate. To estimate the rate and energy efficiency of ClpAP-catalyzed measurements of product sizes, we apply information theory to quantify how precisely the product sizes are controlled. This analysis may also prove to be useful in characterizing the mechanisms of other proteases and nucleases, such as the proteasome and Dicer, which control the sizes of their products.
进行性蛋白酶可以使蛋白质解折叠并将其切割成具有特征尺寸的片段。产物尺寸是如何被控制的详细机制仍存在疑问。控制产物尺寸的一种可能机制是将解折叠的多肽以确定长度的单位转运到蛋白酶活性位点。我们研究了来自大肠杆菌的能量依赖性蛋白酶ClpAP控制其肽产物尺寸的机制。我们发现ClpAP产生的肽产物具有一定的尺寸分布，在肽长度为6 - 8个氨基酸残基处有一个明显的峰值。这种产物尺寸分布与之前在蛋白酶体中观察到的类似，对于干扰转运或蛋白水解的扰动具有抗性。为了解释这些结果，我们提出了一种机制，即转运与蛋白水解交替进行，使得长度或多或少均匀的肽能够从正在转运的底物上逐步切割下来。为了估计ClpAP催化的产物尺寸测量的速率和能量效率，我们应用信息论来量化产物尺寸被控制的精确程度。这种分析可能也有助于表征其他蛋白酶和核酸酶的机制，比如蛋白酶体和Dicer，它们也控制着各自产物的尺寸。
