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Entrance to a lipoxygenase substrate cavity is defined
Author(s) -
Gaffney Betty J.,
Bradshaw Miles,
Freed Jack,
Borbat Peter
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.756.12
Subject(s) - substrate (aquarium) , site directed spin labeling , electron paramagnetic resonance , chemistry , lipoxygenase , crystallography , active site , helix (gastropod) , stereochemistry , enzyme , nuclear magnetic resonance , biochemistry , physics , biology , ecology , snail
A persistent puzzle about lipoxygenase enzymes is how specificity for position and stereochemistry of oxygen addition to polyunsaturated fatty acids is achieved. In lipoxygenase structures, a curved substrate channel approaches the centrally located active site, with ends of the curve near different surface locations. A long‐standing suggestion is that varied specificity results from reversed placement of the polar and methyl ends of the substrate within highly similar protein structures. We approach this problem by placing a spin label on the polar end of a lipoxygenase substrate analog and use paramagnetic distance geometry to locate the substrate spin relative to selected protein positions. Soybean seed lipoxygenase‐1 has been substituted with spin labels at five sites. A five‐point representation of the structure is obtained by measuring ten inter‐site distances by pulsed dipolar EPR spectroscopy (PDS) in doubly spin labeled enzyme constructs. Refinement of spin label coordinates results from modeling and fits to the native protein structure. Finally, the substrate analog polar end is found with five more distance measurements between substrate and protein spins. The spin of the substrate analog is just outside the conjunction of helices‐2 and ‐11. Detailed spin labeling of helix‐2 reveals flexibility permitting entrance to the internal cavity. 1 NIH GM65268 and 2 NIH RR016292