title: Deuterium kinetic isotope effects on the dissociation of a protein-fatty acid complex in the gas phase.
abstract: Deuterium kinetic isotope effects (KIEs) are reported for the first time for the dissociation of a protein-ligand complex in the gas phase. Temperature-dependent rate constants were measured for the loss of neutral ligand from the deprotonated ions of the 1:1 complex of bovine β-lactoglobulin (Lg) and palmitic acid (PA), (Lg + PA)(n-) → Lg(n-) + PA, at the 6- and 7- charge states. At 25 °C, partial or complete deuteration of the acyl chain of PA results in a measurable inverse KIE for both charge states. The magnitude of the KIEs is temperature dependent, and Arrhenius analysis of the rate constants reveals that deuteration of PA results in a decrease in activation energy. In contrast, there is no measurable deuterium KIE for the dissociation of the (Lg + PA) complex in aqueous solution at pH 8. Deuterium KIEs were calculated using conventional transition-state theory with an assumption of a late dissociative transition state (TS), in which the ligand is free of the binding pocket. The vibrational frequencies of deuterated and non-deuterated PA in the gas phase and in various solvents (n-hexane, 1-chlorohexane, acetone, and water) were established computationally. The KIEs calculated from the corresponding differences in zero-point energies account qualitatively for the observation of an inverse KIE but do not account for the magnitude of the KIEs nor their temperature dependence. It is proposed that the dissociation of the (Lg + PA) complex in aqueous solution also proceeds through a late TS in which the acyl chain is extensively hydrated such that there is no significant differential change in the vibrational frequencies along the reaction coordinate and, consequently, no significant KIE.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Lan
authors.initials: L
authors.lastname: Liu
authors.forename: Klaus
authors.initials: K
authors.lastname: Michelsen
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.forename: Paul D
authors.initials: PD
authors.lastname: Schnier
authors.forename: Alex
authors.initials: A
authors.lastname: Brown
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D000818:Animals; D002417:Cattle; D003903:Deuterium; D005740:Gases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D019308:Palmitic Acid / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012997:Solvents / Q000737:chemistry; D013696:Temperature
chemical_list: D005740:Gases; D007782:Lactoglobulins; D012997:Solvents; D019308:Palmitic Acid; D003903:Deuterium