Probing remote residues important for catalysis in Escherichia coli ornithine transcarbamoylase
Probing remote residues important for catalysis in Escherichia coli ornithine transcarbamoylase
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DOI：
10.1371/journal.pone.0228487
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发表时间：
2020-02-06
期刊：
影响因子：
3.7
通讯作者：
Beuning, Penny J.
中科院分区：
文献类型：
--
作者：
Ngu, Lisa;Winters, Jenifer N.;Beuning, Penny J.
了解酶如何获得其巨大的催化能力是生物化学中的一个主要问题。对酶工程应用也需要更多的了解。在许多情况下，酶的效率和特异性取决于不与底物直接接触的残基，称为远程残基。本工作的重点是大肠杆菌鸟氨酸转氨甲酰酶（OTC），它在氨基酸代谢中起着核心作用。据报道，OTC在结合其第一个底物氨甲酰磷酸（CP）后发生诱导拟合构象变化，并且已鉴定出对活性重要的几个残基。使用计算方法的基础上计算的化学性质从理论滴定曲线，基于序列的得分来自进化历史，和蛋白质表面拓扑结构，残基的催化活性的重要预测。这些残基在OTC活性中的作用通过在预测位置构建突变进行了测试，随后进行了稳态动力学测定和变体的底物结合研究。第一层突变R57 A和D231 A、第二层突变H272 L和第三层突变E299 Q导致k（cat）/K-M相对于CP降低57至450倍，相对于鸟氨酸降低44至580倍。第二层突变D140 N和Y160 S也降低了对鸟氨酸的活性。相对于野生型OTC，大多数变体具有降低的稳定性，其中变体H272 L、H272 N和E299 Q具有最大的降低。变体H272 L、E299 Q和R57 A也显示受损的CP结合。除了对催化活性的直接影响外，还观察到对整体蛋白质稳定性和底物结合的影响，这揭示了这些残基如何促进催化的复杂性。
Understanding how enzymes achieve their tremendous catalytic power is a major question in biochemistry. Greater understanding is also needed for enzyme engineering applications. In many cases, enzyme efficiency and specificity depend on residues not in direct contact with the substrate, termed remote residues. This work focuses on Escherichia coli ornithine transcarbamoylase (OTC), which plays a central role in amino acid metabolism. OTC has been reported to undergo an induced-fit conformational change upon binding its first substrate, carbamoyl phosphate (CP), and several residues important for activity have been identified. Using computational methods based on the computed chemical properties from theoretical titration curves, sequence-based scores derived from evolutionary history, and protein surface topology, residues important for catalytic activity were predicted. The roles of these residues in OTC activity were tested by constructing mutations at predicted positions, followed by steady-state kinetics assays and substrate binding studies with the variants. First-layer mutations R57A and D231A, second-layer mutation H272L, and thirdlayer mutation E299Q, result in 57- to 450-fold reductions in k(cat)/K-M with respect to CP and 44- to 580-fold reductions with respect to ornithine. Second-layer mutations D140N and Y160S also reduce activity with respect to ornithine. Most variants had decreased stability relative to wild-type OTC, with variants H272L, H272N, and E299Q having the greatest decreases. Variants H272L, E299Q, and R57A also show compromised CP binding. In addition to direct effects on catalytic activity, effects on overall protein stability and substrate binding were observed that reveal the intricacies of how these residues contribute to catalysis.