Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

Abstract

The enzyme soybean lipoxygenase provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen deuterium exchange experiments to detect a radiating cone of aliphatic side chains that extends from the iron active site of SLO to the protein-solvent interface. Employing eight variants of SLO, nanosecond fluorescence Stokes shifts have been measured using a probe appended to the identified surface loop. We report a remarkable identity of the enthalpies of activation for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within the identified thermal network. While the role of dynamics in enzyme function has been predominantly attributed to a rapidly equilibrating protein conformational landscape, these new data implicate a rapid and cooperative protein quake as the origin of the thermal activation of SLO. A mechanism of catalysis is presented that combines a distributed conformational landscape with a long range and site-specific thermal quake.

Temperature-dependent hydrogen deuterium exchange shows impact of analog binding on adenosine deaminase flexibility but not embedded thermal networks

“…In both instances, opposing protein/solvent interfaces reach inward from the left toward the reactive purine ring of the bound substrate and from the right toward the adjacent active site metal and catalytic base. These findings provide experimental support for the premise of embedded thermal networks within mADA that are largely unchanged upon ligand binding and that can be expected to function in conjunction with distributed protein conformational landscapes that undergo adaptation upon binding of a substrate or relevant analog (See Ref(26) and discussion below).J o u r n a l P r e -p r o o fDiscussionThe multiple levels of information available from HDX experiments. Three tiers of HDX experiments have been described in this study, beginning with single temperature studies of WT and F61A that allow a distinction to be made about the level of protection from ligands that differ 10 6 -fold in their affinities for mADA.…”

“…In both instances, opposing protein/solvent interfaces reach inward from the left toward the reactive purine ring of the bound substrate and from the right toward the adjacent active site metal and catalytic base. These findings provide experimental support for the premise of embedded thermal networks within mADA that are largely unchanged upon ligand binding and that can be expected to function in conjunction with distributed protein conformational landscapes that undergo adaptation upon binding of a substrate or relevant analog (See Ref ( 26 ) and discussion below).…”

“…We note that while both conformational sampling and thermal conduction are expected to occur simultaneously in enzyme reactions, small enthalpic differences among rapidly equilibrating and distributed conformational substates may be expected to distinguish this behavior from the higher enthalpic barriers that are required for ensuing chemical reactions. Ongoing efforts are focused on examining the degree to which the two defined coordinates may be further differentiated using time-dependent spectroscopic measurements ( 26 , 57 , 58 , 59 ).…”

13C ENDOR Spectroscopy-Guided MD Computations Reveals the Structure of the Enzyme-Substrate Complex of an Active, N-linked Glycosylated Lipoxygenase

“…The kinetic behavior of F332A MoLOX is reminiscent of the SLO I553X mutants (X = L or G) that showed little-to-modest changes in kcat, but was accompanied by altered Ea values that are associated with disruption of active site sidechain packing and a network of protein motions. [34][35][36] Finally, we explored alanine mutations at L337 and L522 in MoLOX (Table 4). Kinetic analysis of L337A and L552A show distinct catalytic impacts in MoLOX compared to their homologous mutants, I553A and V750A, 36 in SLO (Table S1).…”

“…[34][35][36] Finally, we explored alanine mutations at L337 and L522 in MoLOX (Table 4). Kinetic analysis of L337A and L552A show distinct catalytic impacts in MoLOX compared to their homologous mutants, I553A and V750A, 36 in SLO (Table S1). Mutation to alanine of L522, which is positioned behind and contacts both L331 and F526, produces a 9-fold decrease in kcat.…”