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

Craig J. Markin, Daniel A. Mokhtari, Siyuan Du, Tzanko Doukov, Fanny Sunden, Jordan A. Cook, Polly M. Fordyce, Daniel Herschlag

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Article numbere2219074120
JournalProceedings of the National Academy of Sciences
Volume120
Issue number29
Early online date10 Jul 2023
DOIs
Publication statusPublished - 18 Jul 2023

Keywords

  • Binding Sites
  • Catalysis
  • Kinetics
  • Mutation
  • Phosphoric Monoester Hydrolases
  • Tungsten Compounds

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