Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13
Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13
复制标题
DOI：
10.1073/pnas.1511328112
复制
发表时间：
2015-07-28
影响因子：
11.1
通讯作者：
Ginsburg, David
中科院分区：
文献类型：
--
作者：
Kretz, Colin A.;Dai, Manhong;Ginsburg, David
蛋白酶在许多生物过程中发挥重要作用，是癌症、炎症和血栓形成的关键介质。然而，目前还缺乏全面和定量的技术来确定蛋白酶的底物特异性。金属蛋白酶ADAMTS13通过切割血管性血友病因子（VWF），降低其促凝活性来调节血液凝固。构建了基于VWF 73个氨基酸片段的诱变底物噬菌体展示文库，并利用高通量测序技术评估了adamts13依赖性文库复杂性随反应时间点的变化。反应速率常数（k(cat)/ k - m）计算了该片段内几乎所有可能的单氨基酸取代。这种大规模平行酶动力学分析详细说明了ADAMTS13的特异性，并证明了P1-P1'底物残基在定义外源性结合域时的关键重要性。这些数据为VWF内的上位倾向提供了经验证据，并显示出与同源物种之间的保护密切相关，突出了VWF的进化选择压力。
Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving vonWillebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (k(cat)/K-M) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1' substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.