Abstract
Heavy-enzyme isotope effects (15N-, 13C-, and 2H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect (kchem light/kchem heavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect (kChem light/kchem heavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steady-state chemistry was slowed 32-fold in F159Y PNP. Pre-steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage.
Original language | English (US) |
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Pages (from-to) | 6456-6461 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 114 |
Issue number | 25 |
DOIs | |
State | Published - Jun 20 2017 |
Keywords
- Enzyme design
- Femtosecond dynamics
- Heavy enzyme
- Purine nucleoside phosphorylase
- Transition path sampling
ASJC Scopus subject areas
- General
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Crystal structure of human purine nucleoside phosphorylase (F159Y) mutant complexed with DADMe-ImmG and phosphate
Harijan, R. K. (Contributor), Zoi, I. (Contributor), Antoniou, D. (Contributor), Schwartz, S. D. (Contributor) & Schramm, V. L. (Contributor), Protein Data Bank (PDB), Jun 21 2017
DOI: 10.2210/pdb5UGF/pdb, https://www.wwpdb.org/pdb?id=pdb_00005ugf
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