Abstract
Understanding gas migration pathways is critical to unraveling structure-function relationships in enzymes that process gaseous substrates such as O2, H2, and N2 This work investigates the role of a defined pathway for O2 in regulating the peroxidation of linoleic acid by soybean lipoxygenase 1. Computational and mutagenesis studies provide strong support for a dominant delivery channel that shuttles molecular oxygen to a specific region of the active site, thereby ensuring the regio- and stereospecificity of product. Analysis of reaction kinetics and product distribution in channel mutants also reveals a plasticity to the gas migration pathway. The findings show that a single site mutation (I553W) limits oxygen accessibility to the active site, greatly increasing the fraction of substrate that reacts with oxygen free in solution. They also show how a neighboring site mutation (L496W) can result in a redirection of oxygen toward an alternate position of the substrate, changing the regio- and stereospecificity of peroxidation. The present data indicate that modest changes in a protein scaffold may modulate the access of small gaseous molecules to enzyme-bound substrates.
Highlights
Transient fluctuations within a protein were proposed to facilitate the delivery of gaseous substrates to their site of binding and/or reactivity
The precise mechanism by which soybean lipoxygenase-1 (SLO-1), as well as other lipoxygenases, maintain the regio- and stereospecificity of product peroxidation has engendered a variety of proposals that include: (i) an alteration in substrate binding as a means of reversing the enantiomeric specificity of O2 addition to a fixed carbon center; (ii) the interchange of a single active site residue between Gly and Ala to alter the position of O2 attack; and (iii) a directed movement of O2 through the protein matrix toward a spatially defined position of substrate that is capable of simultaneously controlling both the position and face of O2 attack on a substrate-derived free radical intermediate [4, 16, 18]
Earlier support for the involvement of this channel came from the generation of an I553F mutant enzyme that showed a reduction in rate for the O2-dependent portion of the SLO-1 reaction accompanied by only a modest impact on the initial C–H abstraction step and the position/stereochemistry of substrate peroxidation [3]
Summary
Study, we have greatly extended this line of inquiry via the incorporation of a range of amino acid side chains into four targeted channel positions. The new findings both corroborate a role for a discrete gas channel in SLO-1 while revealing conditions capable of altering the pathway for O2 delivery to the active site of lipoxygenases
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