caa a22 4500 397555563 CHVBK 20180308164800.0 cr unu---uuuuu 161202e199612 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021545 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021545 Conformational Changes Associated with Activation of Bee Venom Phospholipase A2 [Elektronische Daten] [Tanweer Ahmed, Sharon M. Kelly, Anthony Lawrence, Mokdad Mezna, Nicholas C. Price] 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. Blochem. 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 [Lowrence, 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 α-helix and β-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 fail 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. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence activation nationallicence acylation nationallicence circular dichroism nationallicence conformation nationallicence fluorescence nationallicence phospholipase A2 nationallicence Ahmed Tanweer Protein Crystallography, Joseph Black Building, University of Glasgow Glasgow, G12 8QQ, UK aut Kelly Sharon M. Department of Biological and Molecular Sciences, University of Stirling Stirling, FK9 4LA, UK aut Lawrence Anthony Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow Glasgow, G12 8QQ, UK aut Mezna Mokdad School of Biochemistry, University of Birmingham Birmingham, UK aut Price Nicholas C. Department of Biological and Molecular Sciences, University of Stirling Stirling, FK9 4LA, UK aut The Journal of Biochemistry Oxford University Press 120/6(1996-12), 1224-1231 0021-924X 120:6<1224 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021545 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021545 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ahmed Tanweer Protein Crystallography, Joseph Black Building, University of Glasgow Glasgow, G12 8QQ, UK aut NATIONALLICENCE 700 1- Kelly Sharon M. Department of Biological and Molecular Sciences, University of Stirling Stirling, FK9 4LA, UK aut NATIONALLICENCE 700 1- Lawrence Anthony Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow Glasgow, G12 8QQ, UK aut NATIONALLICENCE 700 1- Mezna Mokdad School of Biochemistry, University of Birmingham Birmingham, UK aut NATIONALLICENCE 700 1- Price Nicholas C. Department of Biological and Molecular Sciences, University of Stirling Stirling, FK9 4LA, UK aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/6(1996-12), 1224-1231 0021-924X 120:6<1224 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford