Extremely elevated room-temperature kinetic isotope effects quantify the critical role of barrier width in enzymatic C-H activation

Shenshen Hu, Sudhir C. Sharma, Alexander D. Scouras, Alexander V. Soudackov, Cody A.Marcus Carr, Sharon Hammes-Schiffer, Tom Alber, Judith P. Klinman

Research output: Contribution to journalArticlepeer-review

Abstract

The enzyme soybean lipoxygenase (SLO) has served as a prototype for hydrogen-tunneling reactions, as a result of its unusual kinetic isotope effects (KIEs) and their temperature dependencies. Using a synergy of kinetic, structural, and theoretical studies, we show how the interplay between donor-acceptor distance and active-site flexibility leads to catalytic behavior previously predicted by quantum tunneling theory. Modification of the size of two hydrophobic residues by site-specific mutagenesis in SLO reduces the reaction rate 104-fold and is accompanied by an enormous and unprecedented room-temperature KIE. Fitting of the kinetic data to a non-adiabatic model implicates an expansion of the active site that cannot be compensated by donor-acceptor distance sampling. A 1.7 Å resolution X-ray structure of the double mutant further indicates an unaltered backbone conformation, almost identical side-chain conformations, and a significantly enlarged active-site cavity. These findings show the compelling property of room-temperature hydrogen tunneling within a biological context and demonstrate the very high sensitivity of such tunneling to barrier width.

Original languageEnglish (US)
Pages (from-to)8157-8160
Number of pages4
JournalJournal of the American Chemical Society
Volume136
Issue number23
DOIs
StatePublished - Jun 11 2014

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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