Biophysical Characterization of a Disabled Double Mutant of Soybean Lipoxygenase: The “Undoing” of Precise Substrate Positioning Relative to Metal Cofactor and an Identified Dynamical Network

Abstract

Soybean lipoxygenase (SLO) has served as a prototype for understanding the molecular origin of enzymatic rate accelerations. The double mutant (DM) L546A/L754A is considered a dramatic outlier, due to the unprecedented size and near temperature-independence of its primary kinetic isotope effect, low catalytic efficiency, and elevated enthalpy of activation. To uncover the physical basis of these features, we herein apply three structural probes: hydrogen-deuterium exchange mass spectrometry, room-temperature X-ray crystallography and EPR spectroscopy on four SLO variants (wild-type (WT) enzyme, DM, and the two parental single mutants, L546A and L754A). DM is found to incorporate features of each parent, with the perturbation at position 546 predominantly influencing thermally activated motions that connect the active site to a proteinsolvent interface, while mutation at position 754 disrupts the ligand field and solvation near the cofactor iron. However, the expanded active site in DM leads to more active site water molecules *

Origins of Enzyme Catalysis: Experimental Findings for C–H Activation, New Models, and Their Relevance to Prevailing Theoretical Constructs

“…Studies under investigation suggest that the rate impairment may reflect an inability of the substrate to position itself properly in relation to catalytically-relevant protein motions. 56…”

EPR Spectroscopic Studies of Lipoxygenases

“…Shifts in His‐to‐Fe 3+ charge‐transfer optical bands (400–425 nm region) in first‐coordination sphere mutants of SBL1 also report on reduction potentials. Reduction potentials correlate well with factors affecting k cat of lipoxygenase first‐coordination sphere variants, but less well for mutation at more distant sites …”

“…Reduction potentials correlate well with factorsaffecting k cat of lipoxygenase first-coordination sphere variants, [33] but less well for mutation at more distant sites. [34] Some of the human lipoxygenases have also been examined by EPR to test cycling of the iron oxidation state in mechanistic steps. Human 5-lipoxygenase (5-LO) oxidizes arachidonic acid at carbon-5 as af irst step in leukotriene synthesis.…”

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

“…A few years later, a high-resolution (1.4 Å) structural model of SLO-1 was reported [ 24 ], with the putative substrate portal proposed to be gated by residues T259 and K260 on helix 2 and L541 on the arched helix. Since then, dozens of SLO-1 crystal structures have been solved (see references [ 21 , 25 , 26 , 27 , 28 ], for example), though all are devoid of substrate (analogs) or allosteric effectors. Another isoform, SLO-3, was resolved with the product 13 S -hydroperoxyoctadecadienoic acid (13 S -HpOD) filling the substrate pocket [ 29 ].…”

“…100-fold decrease in catalytic proficiency in either case [43,44]. These mutations do not greatly change the redox potential of the metal center, but perturb the proper positioning of substrate binding with respect to the catalytic center [28,44].…”