Conformational changes associated with activation of bee venom phospholipase A2.
Bee venom PLA2 possesses a binding site for long-chain fatty acids that can be acylated by long-chain fatty acid imidazolides [Drainas, D. and Lawrence, A.J. (1978) Eur. J. Biochem. 91, 131-138]. Occupation of the site either by oleic acid or the oleoyl residue enhances the catalytic activity by 45.7-fold in the presence of 20% 1-propanol and occupation of the site by the oleoyl residue increases the lytic activity against rabbit erythrocytes by 60-fold. Treatment of the enzyme with oleic acid and glutaraldehyde is known to produce irreversible activation [Lawrence, A.J. and Moores, G.R. (1975) FEBS Lett. 49, 287-291]. Here we show that reduction of the glutaraldehyde-treated enzyme with borohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activation does not require the presence of oleic acid and indicating that activation is due to a change in the conformation rather than the hydrophobicity of the protein. A kinetic study of enzyme activated by oleoyl imidazolide showed that this modification stabilizes the protein against reversible inactivation by 1-propanol. Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in secondary 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 fall attributed to occupation of the binding site followed by a progressive decrease with a shift of the emission maximum from 341 to 348 nm. The rate of the second phase closely matched the rate of increase in catalytic activity of the enzyme. Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change. These results support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site increases the exposure of one or both of the Trp residues to the aqueous environment. Binding studies show that activation by oleoyl imidazolide does not increase the affinity of the enzyme for the erythrocyte membrane. It is proposed that occupation of a long-chain fatty acid binding site on the enzyme enhances catalytic activity by changing the conformation of the protein rather than acting as a hydrophobic anchor to the substrate surface.
Ahmed
T
T
University of Glasgow, UK.
Kelly
S M
SM
Lawrence
A J
AJ
Mezna
M
M
Price
N C
NC
D000433:1-Propanol / Q000494:pharmacology; D000215:Acylation; D000818:Animals; D001514:Bee Venoms / Q000737:chemistry*; D002942:Circular Dichroism; D004789:Enzyme Activation; D004912:Erythrocytes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000378:metabolism; D019301:Oleic Acid / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2; D011487:Protein Conformation; D011817:Rabbits
D001514:Bee Venoms; D007093:Imidazoles; D019301:Oleic Acid; C018353:oleoylimidazolide; D000433:1-Propanol; D010741:Phospholipases A; D054467:Phospholipases A2