title: Extremely elevated room-temperature kinetic isotope effects quantify the critical role of barrier width in enzymatic C-H activation.
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 10(4)-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.
authors.affiliation: Department of Chemistry, University of California , Berkeley, California 94720, United States.
authors.forename: Shenshen
authors.initials: S
authors.lastname: Hu
authors.forename: Sudhir C
authors.initials: SC
authors.lastname: Sharma
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Scouras
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Soudackov
authors.forename: Cody A Marcus
authors.initials: CA
authors.lastname: Carr
authors.forename: Sharon
authors.initials: S
authors.lastname: Hammes-Schiffer
authors.forename: Tom
authors.initials: T
authors.lastname: Alber
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D001665:Binding Sites; D002244:Carbon / Q000737:chemistry*; D002384:Catalysis; D003198:Computer Simulation; D004579:Electron Transport; D004789:Enzyme Activation; D006859:Hydrogen / Q000737:chemistry*; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D008041:Linoleic Acids / Q000737:chemistry; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011789:Quantum Theory; D013025:Glycine max / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007554:Isotopes; D008041:Linoleic Acids; D002244:Carbon; D006859:Hydrogen; D008084:Lipoxygenase