Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C−H Activation by Soybean Lipoxygenase‐1

Abstract

The ability to relate a hierarchy of protein motions to function remains a compelling experimental challenge at the interface of chemistry and biology. In particular, the proposed contribution of distinctly different classes of local vs. global protein motions during enzymatic catalysis of bond making/breaking processes has been difficult to capture and verify. Herein we employ soybean lipoxygenase-1 as a model system to investigate the impact of high pressure at variable temperatures on the hydrogen tunneling properties of wild type protein and three single site mutants. For all variants, pressure dramatically elevates the experimental enthalpies of activation accompanying the C-H activation step, as predicted for non-physiological conditions that lead to impairment of a protein’s global conformational landscape. In marked contrast, the primary kinetic isotope effects 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, vs. substrate kinetic isotope effects provides direct experimental verification of two classes of protein motions: local, isotope-dependent donor-acceptor distance sampling modes that are distinct from the more global, isotope independent search for productive protein conformational sub-states.

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis

“…Numerically fitting these data then gives Δ H ‡ , Δ S ‡ , Δ G ‡ , , Δ V ‡ , Δβ ‡ and Δα ‡ reflecting the changes in enthalpy, entropy, Gibbs free energy, heat capacity, activation volume, compressibility and expansivity between the enzyme–substrate complex and the enzyme–transition state complex respectively. The enzyme kinetics for only a relatively few enzymes has been treated by combined p / T studies . In these cases, the p /T plane has not been fitted with a numerical model meaning at least and ∆α ‡ are not determined.…”

Enhanced Rigidification within a Double Mutant of Soybean Lipoxygenase Provides Experimental Support for Vibronically Nonadiabatic Proton-Coupled Electron Transfer Models

“… 19 , 30 Recently, SLO has also served as an excellent system in which to interrogate the relationship between multidimensional conformational sampling and hydrogen tunneling. 31 − 33…”

“…We next investigated the status of global conformational sampling in DM-SLO by examining the combined impact of temperature and pressure on the observed rates of DM-SLO toward H-LA as substrate. Using established protocols for the WT SLO and three single site mutants (section 1.5 in SI ), 31 the resulting data can be evaluated in the context of a pressure parameter S , which is the ratio of the k cat-H at each elevated pressure to k cat-H at ambient pressure ( Table 2 ). At 288 K, with the exception of WT, all of the rate constants for H-LA increase with pressure, and this effect tends to be more pronounced for the mutants that contain L754A.…”

The Mechanism of Sugar C−H Bond Oxidation by a Flavoprotein Oxidase Occurs by a Hydride Transfer Before Proton Abstraction

“…[73] The protein motions that affect catalytic enhancement wasa lso seen on hydrostatic pressure studies involving the reductive half-reaction of morphinone reductase, [90] pentaerythritol tetranitrate reductase, [91] aromatic amine dehydrogenase, [92] and soybean lipoxygenase-1. [93] The mechanism in Model 3c an also explain the mechanistic results of other enzymes in the GMC family.C holine oxidase exhibits ap rimary deuterium KIE in the flavin reduction step, suggesting that flavin reductioni sr ate limiting. [88] No solvent deuterium KIE (SKIE) was observed with this enzyme.…”

“…The mechanism of C−H bond oxidation in Model 3 can also overcome difficulties of the deprotonation of the sugar O−H proton, which typically has a p K a around 18–19 . The protein motions that affect catalytic enhancement was also seen on hydrostatic pressure studies involving the reductive half‐reaction of morphinone reductase, pentaerythritol tetranitrate reductase, aromatic amine dehydrogenase, and soybean lipoxygenase‐1 …”