Detecting and Characterizing the Kinetic Activation of Thermal Networks in Proteins: Thermal Transfer from a Distal, Solvent-Exposed Loop to the Active Site in Soybean Lipoxygenase

Abstract

The rate-limiting chemical reaction catalyzed by soybean lipoxygenase (SLO) involves quantum mechanical tunneling of a hydrogen atom from substrate to its active site ferric-hydroxide cofactor. SLO has emerged as a prototypical system for linking the thermal activation of a protein scaffold to the efficiency of active site chemistry. Significantly, hydrogen–deuterium exchange-mass spectrometry (HDX-MS) experiments on wild type and mutant forms of SLO have uncovered trends in the enthalpic barriers for HDX within a solvent-exposed loop (positions 317–334) that correlate well with trends in the corresponding enthalpic barriers for k cat. A model for this behavior posits that collisions between water and loop 317–334 initiate thermal activation at the protein surface that is then propagated 15–34 Å inward toward the reactive carbon of substrate in proximity to the iron catalyst. In this study, we have prepared protein samples containing cysteine residues either at the tip of the loop 317–334 (Q322C) or on a control loop, 586–603 (S596C). Chemical modification of cysteines with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (Badan, BD) provides site-specific probes for the measurement of fluorescence relaxation lifetimes and Stokes shift decays as a function of temperature. Computational studies indicate that surface water structure is likely to be largely preserved in each sample. While both loops exhibit temperature-independent fluorescence relaxation lifetimes as do the Stokes shifts for S596C–BD, the activation enthalpy for the nanosecond solvent reorganization at Q322C–BD (E a(k solv) = 2.8(0.9) kcal/mol)) approximates the enthalpy of activation for catalytic C–H activation (E a(k cat) = 2.3(0.4) kcal/mol). This study establishes and validates the methodology for measuring rates of rapid local motions at the protein/solvent interface of SLO. These new findings, when combined with previously published correlations between protein motions and the rate-limiting hydride transfer in a thermophilic alcohol dehydrogenase, provide experimental evidence for thermally induced “protein quakes” as the origin of enthalpic barriers in catalysis.

Fatty Acid Allosteric Regulation of C-H Activation in Plant and Animal Lipoxygenases

“…3.1 Å, consistent with the expectation of van der Waals contacts [21]. The reduction in this distance thus occurs transiently and may be facilitated by defined protein conformational networks involved in thermal activation [27,94]. The efficiency of active site compaction is influenced by proper steric bulk of hydrophobic residues at the active site [26,43,95,96].…”

Hydrogen deuterium exchange defines catalytically linked regions of protein flexibility in the catechol O -methyltransferase reaction

“…Specifically, through the application of site-specific mutagenesis, time-dependent fluorescence spectroscopic methods, and HDX-MS, two distinct long-range dynamical networks have been identified that connect solvent exposed regions of protein to the substrate binding domain (26); additionally, enthalpic barriers from kinetic measurements of Stokes shifts and T-jump FRET are seen to be identical to catalysis (15,17,27). In recent studies of a very different monomeric, single-substrate enzyme (soybean lipoxygenase), HDX-MS results link the thermal activation of a solvent-exposed loop to the positioning of the buried reactive C-H bond of substrate 15 to 30 Å away (28), once again with an enthalpic barrier for catalysis that is mirrored in Stokes shifts of a solvent-exposed chromophore (29). The goal of the present work was to extend these developed methodologies to other types of reaction, specifically the well-studied methyl transfer catalyzed by COMT (33,34,37).…”

“…We have previously demonstrated that performing HDX as a function of temperature can uncover the regions within a protein that dominate the thermal activation of active site chemistry. In the case of C-H activating enzymes such as the thermophilic alcohol dehydrogenase (ht-ADH) (25)(26)(27) and soybean lipoxygenase (SLO) (28,29), where quantum mechanical tunneling dominates the chemical coordinate, in-depth kinetic and biophysical probes have revealed a model for catalysis in which the thermal activation barrier to reaction comes from the protein scaffold via wellresolved networks that connect solvent-exposed surfaces to the enzyme active site (26)(27)(28)(29)(30)(31).…”

Energy transfer to the phonons of a macromolecule through light pumping

“…Redox reactions and mitochondria produce weak UV photons that might excite Tryptophan and Tyrosine amino acids 21 , 22 in proteins, as well nucleotides of DNA and RNA. Also an anisotropic momentum transfer operated by water molecules or ions could make the job 23 . In either cases of metabolically generated photons or of ion collisions (phosphate stemming from ATP hydrolysis or other) we can assume that the external energy input for a biomolecule occurs through the generation of “hot points”, as in the case of light activated fluorophores, and mediated by either radiative or collisional electronic excitation.…”