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.
The enzyme soybean lipoxygenase (SLO) 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 define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein–solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (
E
a
) for the Stokes shifts decay rates and the millisecond C–H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.
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.
Artificial metalloproteins
(ArMs) have recently gained significant
interest due to their potential to address issues in a broad scope
of applications, including biocatalysis, biotechnology, protein assembly,
and model chemistry. ArMs are assembled by the incorporation of a
non-native metallocofactor into a protein scaffold. This can be achieved
by a number of methods that apply tools of chemical biology, computational
de novo
design, and synthetic chemistry. In this Perspective,
we highlight select systems in the hope of demonstrating the breadth
of ArM design strategies and applications and emphasize how these
systems address problems that are otherwise difficult to do so with
strictly biochemical or synthetic approaches.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.