title: Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C-H Activation by Soybean Lipoxygenase-1.
abstract: The proposed contributions of distinct classes of local versus global protein motions during enzymatic bond making/breaking processes has been difficult to verify. We employed soybean lipoxygenase-1 as a model system to investigate the impact of high pressure at variable temperatures on the hydrogen-tunneling properties of the wild-type protein and three single-site mutants. For all variants, pressure dramatically elevates the enthalpies of activation for the C-H activation. In contrast, the primary kinetic isotope effects (KIEs) for C-H activation and their corresponding temperature dependencies remain unchanged up to ca. 700 bar. The differential impact of elevated hydrostatic pressure on the temperature dependencies of rate constants versus substrate KIEs provides direct evidence for two distinct classes of protein motions: local, isotope-dependent donor-acceptor distance-sampling modes, and a more global, isotope-independent search for productive protein conformational sub-states.
authors.affiliation: Department of Chemistry, University of California, California Institute for Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA.
authors.forename: Shenshen
authors.initials: S
authors.lastname: Hu
authors.affiliation: Department of Chemistry, University of California, California Institute for Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA.|Department of Biochemistry, UW Box 357350, 1705 NE Pacific St., Seattle, WA, 98195-7350, USA.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Cattin-Ortolá
authors.affiliation: Department of Chemistry, University of California, California Institute for Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA.|DuPont Industrial Biosciences, 925 Page Mill Rd, Palo Alto, CA, 94304, USA.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Munos
authors.affiliation: Department of Chemistry, University of California, California Institute for Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA. klinman@berkeley.edu.|Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA. klinman@berkeley.edu.
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D006874:Hydrostatic Pressure; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013696:Temperature
chemical_list: C101084:lipoxygenase L-1; D008084:Lipoxygenase
keywords: biocatalysis; enzymes; hydrostatic pressure; protein conformations; protein motions