Catalysis sensitive conformational changes in soybean lipoxygenase revealed by limited proteolysis and monoclonal antibody experiments.
Soybean lipoxygenases catalyze lipid hydroperoxidation of polyunsaturated fatty acids. Putative ligand mediated conformational changes in soybean lipoxygenase 3 (L3) were studied by a combination of limited proteolysis and a series of monoclonal antibodies that recognize discontinuous epitopes and alter catalysis (inhibition and activation). Trypsin cleaved L3 (97 kDa) into C-terminal 60 kDa and N-terminal 37 kDa fragments. The 37 kDa fragment was obtained from a 38 kDa fragment formed initially. Using protein footprinting, the epitopes of the antibodies were mapped to the 37 kDa fragment. Proteolysis in the presence of a substrate analog inhibitor, oleic acid, generated the 60 and the 38 kDa fragments only. No further proteolysis of the 38 kDa fragment was seen even after prolonged incubation. This was not a detergent effect since the altered proteolysis was not obtained in the presence of SDS or Tween 20. Binding of a monoclonal antibody to L3 in the presence of oleic acid was substantially reduced providing additional evidence for a conformational change induced by the oleic acid-lipoxygenase interaction. These observations are interpreted using the recently solved three-dimensional structure of L3. It is apparent that while the protein is composed of a small N-terminal beta-barrel domain and a large principally alpha-helical C-terminal domain, proteolysis does not take place at a linking region between the two domains. The proteolysis results makes it clear that the smaller domain is connected across the entire length of the larger domain to a narrow, tongue-like projection that extends into the vicinity of the entrance to the proposed substrate binding channel.(ABSTRACT TRUNCATED AT 250 WORDS)
Ramachandran
S
S
Department of Chemistry, University of Toledo, Ohio 43606, USA.
Richards-Sucheck
T J
TJ
Skrzypczak-Jankun
E
E
Wheelock
M J
MJ
Funk
M O
MO
D000911:Antibodies, Monoclonal / Q000276:immunology; D002384:Catalysis; D003196:Computer Graphics; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D007527:Isoenzymes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D009829:Oleic Acids; D010446:Peptide Fragments; D019301:Oleic Acid; D008084:Lipoxygenase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin