Interfacial Mechanism of Phospholipase A2: pH-Dependent Inhibition and Me-β-cyclodextrin Activation

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• 作者：Hanna P. Wacklin
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：June 30, 2009
• 年：2009
• 卷：48
• 期：25
• 页码：5874-5881
• 全文大小：822K
• 年卷期：v.48,no.25(June 30, 2009)
• ISSN：1520-4995

文摘

The pH-dependent activity of phospholipase A₂ (PLA₂) from Naja mossambica mossambica venom and the membrane-water partitioning of the lipid hydrolysis products were investigated in solid-supported palmitoyl-oleyl-phosphatidylcholine-d₃₁ (POPC-d₃₁) membranes using neutron reflection. At pH 5, PLA₂ interacts only weakly with the substrate membrane and leads to no observable membrane breakdown, which is consistent with protonation of the catalytic histidine (His48, pK_a 6.2). The rate of the lyso-lipid partitioning into the solution phase is the same at pH 9 as at pH 7.4, and the relative membrane-water partitioning of the products is essentially the same; that is, the fatty acid accumulates in the membrane, and only the lyso-lipid is solubilized. However, Me-β-cyclodextrin (Me-β-CD) activates PLA₂ irrespective of pH by facilitating the solubilization of the lyso-lipid product, but not the fatty acid, of which only 22% is encapsulated at pH 9. Since no product solubilization is observed at pH 5 in the absence of Me-β-CD, this suggests that the hydrolytic mechanism of PLA₂ is not fully disabled at pH 5 but is inhibited by a mechanism, which is counteracted by Me-β-CD-mediated release of the lyso-lipid. Me-β-CD does not interact with the substrate membrane, which indicates that at low pH the product extraction occurs directly from the enzyme active site outside the immediate membrane−water interface, whereas at pH 7−9, direct solubilization of the lyso-lipid from the membrane can also contribute to activation of PLA₂.