CATALYSIS SENSITIVE CONFORMATIONAL-CHANGES IN SOYBEAN LIPOXYGENASE REVEALED BY LIMITED PROTEOLYSIS AND MONOCLONAL-ANTIBODY EXPERIMENTS

Soybean lipoxygenases catalyze lipid hydroperoxidation of polyunsatura ted fatty acids. Putative ligand mediated conformational changes in so ybean lipoxygenase 3 (L3) were studied by a combination of Limited pro teolysis and a series of monoclonal antibodies that recognize disconti nuous epitopes and alter catalysis (inhibition and activation). Trypsi n cleaved L3 (97 kDa) into C-terminal 60 kDa and N-terminal 37 kDa fra gments. 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 kD a fragments only. No further proteolysis of the 38 kDa fragment was se en even after prolonged incubation This was not a detergent effect sin ce the altered proteolysis pattern was not obtained in the presence of SDS or Tween 20. Binding of a monoclonal antibody to L3 in the presen ce of oleic acid was substantially reduced providing additional eviden ce for a conformational change induced by the oleic acid-lipoxygenase interaction. These observations are interpreted using the recently sol ved three-dimensional structure of L3. It is apparent that while the p rotein is composed of a small N-terminal beta-barrel domain and a larg e principally alpha-helical C-terminal domain, proteolysis does not ta ke place at a linking region between the two domains. The proteolysis result 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 substr ate binding channel. It is proposed that conformational changes take p lace upon oleic acid binding which are transmitted through the protein and alter structural features in the N-terminal fragment that are sen sitive to proteolysis and antibody binding. This is the first direct e vidence for conformational changes related to catalysis in lipoxygenas e.