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Photo Lynn Kamerlin

Lynn Kamerlin

Professor

Photo Lynn Kamerlin

Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase

Author

  • Martin Pfeiffer
  • Rory M Crean
  • Catia Moreira
  • Antonietta Parracino
  • Gustav Oberdorfer
  • Lothar Brecker
  • Friedrich Hammerschmidt
  • Shina Caroline Lynn Kamerlin
  • Bernd Nidetzky

Summary, in English

The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical, and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is ∼104-fold. Cooperativity with the catalytic acid adds ≥102-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.

Publishing year

2022-03-18

Language

English

Pages

3357-3370

Publication/Series

ACS Catalysis

Volume

12

Issue

6

Document type

Journal article

Publisher

The American Chemical Society (ACS)

Status

Published

ISBN/ISSN/Other

  • ISSN: 2155-5435