Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase

Rajesh K. Harijan, Ioanna Zoi, Dimitri Antoniou, Steven D. Schwartz, Vern L. Schramm

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)6456-6461
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number25
DOIs
StatePublished - Jun 20 2017

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Purine-Nucleoside Phosphorylase
Isotopes
Catalytic Domain
Enzymes
Light
Catalysis
Proteins

Keywords

  • Enzyme design
  • Femtosecond dynamics
  • Heavy enzyme
  • Purine nucleoside phosphorylase
  • Transition path sampling

ASJC Scopus subject areas

  • General

Cite this

Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase. / Harijan, Rajesh K.; Zoi, Ioanna; Antoniou, Dimitri; Schwartz, Steven D.; Schramm, Vern L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 114, No. 25, 20.06.2017, p. 6456-6461.

Research output: Contribution to journalArticle

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