Decoupling of catalysis and transition state analog binding from mutations throughout a phosphatase revealed by high-throughput enzymology

活动站点 过渡态模拟 化学 过渡状态 结合位点 酶催化 立体化学 磷酸酶 变构调节 催化作用 生物物理学 生物化学 生物
作者
Craig J. Markin,Daniel A. Mokhtari,Siyuan Du,Tzanko Doukov,Fanny Sunden,Jordan A. Cook,Polly M. Fordyce,Daniel Herschlag
出处
期刊:Proceedings of the National Academy of Sciences of the United States of America [Proceedings of the National Academy of Sciences]
卷期号:120 (29) 被引量:1
标识
DOI:10.1073/pnas.2219074120
摘要

Using high-throughput microfluidic enzyme kinetics (HT-MEK), we measured over 9,000 inhibition curves detailing impacts of 1,004 single-site mutations throughout the alkaline phosphatase PafA on binding affinity for two transition state analogs (TSAs), vanadate and tungstate. As predicted by catalytic models invoking transition state complementary, mutations to active site and active-site-contacting residues had highly similar impacts on catalysis and TSA binding. Unexpectedly, most mutations to more distal residues that reduced catalysis had little or no impact on TSA binding and many even increased tungstate affinity. These disparate effects can be accounted for by a model in which distal mutations alter the enzyme’s conformational landscape, increasing the occupancy of microstates that are catalytically less effective but better able to accommodate larger transition state analogs. In support of this ensemble model, glycine substitutions (rather than valine) were more likely to increase tungstate affinity (but not more likely to impact catalysis), presumably due to increased conformational flexibility that allows previously disfavored microstates to increase in occupancy. These results indicate that residues throughout an enzyme provide specificity for the transition state and discriminate against analogs that are larger only by tenths of an Ångström. Thus, engineering enzymes that rival the most powerful natural enzymes will likely require consideration of distal residues that shape the enzyme’s conformational landscape and fine-tune active-site residues. Biologically, the evolution of extensive communication between the active site and remote residues to aid catalysis may have provided the foundation for allostery to make it a highly evolvable trait.
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