title: Kinetic Detection of Orthogonal Protein and Chemical Coordinates in Enzyme Catalysis: Double Mutants of Soybean Lipoxygenase.
abstract: Soybean lipoxygenase-1 (SLO-1) is a paradigmatic enzyme system for studying the contribution of hydrogen tunneling to enzymatic proton-coupled electron transfer processes. In this study, the impact of pairs of double mutants on the properties of SLO-1 is presented. Steady-state rates and their deuterium kinetic isotope effects (KIEs) have been measured for the bimolecular reaction of enzyme with free substrate (kcat/Km) and compared to the unimolecular rate constant, kcat. A key kinetic finding is that the competitive KIEs on the second-order rate constant (kcat/Km) are all reduced from (D)kcat and, despite large changes in rate and activation parameters, remain essentially unaltered under a variety of conditions. These data implicate a protein reaction coordinate that is orthogonal to the chemical reaction coordinate and controls the concentration of the active enzyme. This study introduces a new means to interrogate the alteration of conformational landscapes that can occur following site-specific mutagenesis.
authors.affiliation: Department of Chemistry, ‡Department of Molecular and Cell Biology, and California Institute for Quantitative Biosciences, University of California , Berkeley, California 94720, United States.
authors.forename: Sudhir C
authors.initials: SC
authors.lastname: Sharma
authors.affiliation: Department of Chemistry, ‡Department of Molecular and Cell Biology, and California Institute for Quantitative Biosciences, University of California , Berkeley, California 94720, United States.
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D002384:Catalysis; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000502:physiology*; D013025:Glycine max / Q000201:enzymology*
chemical_list: C101084:lipoxygenase L-1; D008084:Lipoxygenase