Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

Rafael G. Silva, Andrew S. Murkin, Vern L. Schramm

Research output: Contribution to journalArticlepeer-review

100 Scopus citations


Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nucleoside phosphorylase (PNP) forms a ribocation-like transition state in the phosphorolysis of purine nucleosides and fast protein motions have been proposed to participate in barrier crossing. In the present study, 13C-, 15N-, 2H-labeled human PNP (heavy PNP) was expressed, purified to homogeneity, and shown to exhibit a 9.9% increase in molecular mass relative to its unlabeled counterpart (light PNP). Kinetic isotope effects and steady-state kinetic parameters were indistinguishable for both enzymes, indicating that transition-state structure, equilibrium binding steps, and the rate of product release were not affected by increased protein mass. Single-turnover rate constants were slowed for heavy PNP, demonstrating reduced probability of chemical barrier crossing from enzyme-bound substrates to enzyme-bound products. In a second, independent method to probe barrier crossing, heavy PNP exhibited decreased forward commitment factors, also revealing mass-dependent decreased probability for barrier crossing. Increased atomic mass in human PNP alters bond vibrational modes on the femtosecond time scale and reduces on-enzyme chemical barrier crossing. This study demonstrates coupling of enzymatic bond vibrations on the femtosecond time scale to barrier crossing.

Original languageEnglish (US)
Pages (from-to)18661-18665
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number46
StatePublished - Nov 15 2011


  • Enzymatic catalysis
  • Femtosecond motions
  • Kinetic isotope effect
  • Protein dynamics
  • Transition state structure

ASJC Scopus subject areas

  • General


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