Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers

The oxidation of polyunsaturated fatty acids by lipoxygenases
(LOXs)
is initiated by a C–H cleavage step in which the hydrogen atom
is transferred quantum mechanically (i.e., via tunneling). In these
reactions, protein thermal motions facilitate the conversion of ground-state
enzyme–substrate complexes to tunneling-ready configurations
and are thus important for transferring energy from the solvent to
the active site for the activation of catalysis. In this report, we
employed temperature-dependent hydrogen–deuterium exchange
mass spectrometry (TDHDX-MS) to identify catalytically linked, thermally
activated peptides in a representative animal LOX, human epithelial
15-LOX-2. TDHDX-MS of wild-type 15-LOX-2 was compared to two active
site mutations that retain structural stability but have increased
activation energies (Ea) of catalysis.
The Ea value of one variant, V427L, is
implicated to arise from suboptimal substrate positioning by increased
active-site side chain rotamer dynamics, as determined by X-ray crystallography
and ensemble refinement. The resolved thermal network from the comparative Eas of TDHDX-MS between wild-type and V426A is
localized along the front face of the 15-LOX-2 catalytic domain. The
network contains a clustering of isoleucine, leucine, and valine side
chains within the helical peptides. This thermal network of 15-LOX-2
is different in location, area, and backbone structure compared to
a model plant lipoxygenase from soybean that exhibits a low Ea value of catalysis compared to the human ortholog.
The presented data provide insights into the divergence of thermally
activated protein motions in plant and animal LOXs and their relationships
to the enthalpic barriers for facilitating hydrogen tunneling.