Fishovitz Jennifer, Sha Zhou, Chilakala Sujatha, Cheng Iteen, Xu Yan, Lee Irene
Department of Chemistry and Physics, Saint Mary's CollegeNotre Dame, IN, United States.
Department of Chemistry, Case Western Reserve UniversityCleveland, OH, United States.
Front Mol Biosci. 2017 Jul 11;4:47. doi: 10.3389/fmolb.2017.00047. eCollection 2017.
Lon, also known as Protease La, is one of the simplest ATP-dependent proteases. It is a homooligomeric enzyme comprised of an ATPase domain and a proteolytic domain in each enzyme subunit. Despite sharing about 40% sequence identity, human and Lon proteases utilize a highly conserved ATPase domain found in the AAA+ family to catalyze ATP hydrolysis, which is needed to activate protein degradation. In this study, we utilized mechanistic enzymology techniques to show that despite comparable k and K parameters found in the ATPase activity, human and Lon exhibit significantly different susceptibility to ADP inhibition. Due to the low affinity of human Lon for ADP, the conformational changes in human Lon generated from the ATPase cycle are also different. The relatively low affinity of human Lon for ADP cannot be accounted for by reversibility in ATP hydrolysis, as a positional isotope exchange experiment demonstrated both Lon and human Lon catalyzed ATP hydrolysis irreversibly. A limited tryptic digestion study however indicated that human and Lon bind to ADP differently. Taken together, the findings reported in this research article suggest that human Lon is not regulated by a substrate-promoted ADP/ATP exchange mechanism as found in the bacterial enzyme homolog. The drastic difference in structural changes associated with ADP interaction with the two protease homologs offer potential for selective inhibitor design and development through targeting the ATPase sites. In addition to revealing unique mechanistic differences that distinguish human vs. bacterial Lon, this article underscores the benefit of mechanistic enzymology in deciphering the physiological mechanism of action of Lon proteases and perhaps other closely related ATP-dependent proteases in the future.
Lon，也被称为蛋白酶La，是最简单的ATP依赖性蛋白酶之一。它是一种同寡聚酶，每个酶亚基都由一个ATP酶结构域和一个蛋白水解结构域组成。尽管人类和Lon蛋白酶的序列同一性约为40%，但它们利用AAA+家族中高度保守的ATP酶结构域来催化ATP水解，这是激活蛋白质降解所必需的。在本研究中，我们利用机制酶学技术表明，尽管在ATP酶活性中发现了相当的k和K参数，但人类和Lon对ADP抑制的敏感性却有显著差异。由于人类Lon对ADP的亲和力较低，ATP酶循环产生的人类Lon的构象变化也不同。人类Lon对ADP的相对低亲和力不能用ATP水解的可逆性来解释，因为位置同位素交换实验表明Lon和人类Lon都不可逆地催化ATP水解。然而，一项有限的胰蛋白酶消化研究表明，人类和Lon与ADP的结合方式不同。综上所述，本研究文章报道的结果表明，人类Lon不像细菌酶同源物那样受底物促进的ADP/ATP交换机制调节。与ADP与两种蛋白酶同源物相互作用相关的结构变化的巨大差异为通过靶向ATP酶位点进行选择性抑制剂的设计和开发提供了潜力。除了揭示区分人类与细菌Lon的独特机制差异外，本文还强调了机制酶学在未来解读Lon蛋白酶以及可能其他密切相关的ATP依赖性蛋白酶的生理作用机制方面的益处。
