CONFORMATIONAL-CHANGES ASSOCIATED WITH ACTIVATION OF BEE VENOM PHOSPHOLIPASE A(2)

Bee venom PLA, possesses a binding site for long-chain fatty acids tha t can be acylated by long-chain fatty acid imidazolides [Drainas, D, a nd Lawrence, A,J, (1978) fur. J, Biochem, 91, 131-138], Occupation of the site either by oleic acid or the oleoyl residue enhances the catal ytic activity by 45.7-fold in the presence of 20% l-propanol and occup ation of the site by the oleoyl residue increases the lyric activity a gainst rabbit erythrocytes by 60-fold, Treatment of the enzyme with ol eic acid and glutaraldehyde is known to produce irreversible activatio n [Lawrence, A,J, and Moores, G.R. (1975) FEES Lett, 49, 287-291], Her e we show that reduction of the glutaraldehyde-treated enzyme with bor ohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activatio n does not require the presence of oleic acid and indicating that acti vation is due to a change in the conformation rather than the hydropho bicity of the protein, A kinetic study of enzyme activated by oleoyl i midazolide showed that this modification stabilizes the protein agains t reversible inactivation by 1-propanol, Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in se condary structure with apparent increase in both alpha-helix and beta- sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with an initial fal l attributed to occupation of the binding site followed by a progressi ve decrease with a shift of the emission maximum from 341 to 348 nm. T he rate of the second phase closely matched the rate of increase in ca talytic activity of the enzyme, Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change, These resul ts support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site inc reases the exposure of one or both of the Trp residues to the aqueous environment, Binding studies show that activation by oleoyl imidazolid e does not increase the affinity of the enzyme for the erythrocyte mem brane. It is proposed that occupation of a long- chain fatty acid bind ing site on the enzyme enhances catalytic activity by changing the con formation of the protein rather than acting as a hydrophobic anchor to the substrate surface.