We have recently shown that an endogenous phospholipase A2 from bovine erythrocytes does not hydrolyse NAPEs (N-acyl l-α-phosphatidylethanolamines), which accumulate remarkably in this system [Florin-Christensen, Suarez, Florin-Christensen, Wainszelbaum, Brown, McElwain and Palmer (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 7736–7741]. Here we investigate the causes underlying this resistance. N-acylation of PE (l-α-phosphatidylethanolamine) results in alteration of charge, head-group volume and conformation, the last two features depending on the N-acyl chain length. To evaluate each effect separately, we synthesized NAPEs with selected N-acyl chain length. We found that phospholipase A2 has considerable activity against N-acetyl PE, but is poorly active against N-butanoyl PE and only marginally active against N-hexanoyl PE, whereas the activity is completely lost when N-hexadecanoyl PE is presented as a substrate. On the other hand, N-hexanoyl PE does not inhibit phospholipase A2 activity, suggesting that this substrate fails to enter the hydrophobic channel. Phospholipase C presents a similar, but less sharp pattern. Molecular dynamics simulations of the polar head group of selected NAPEs reveal a substantially increased conformational variability as the N-acyl chain grows. This larger conformational space represents an increased impairment limiting the access of these molecules to the active site. Our data indicate that, whereas a change in charge contributes to diminished activity, the most relevant effects come from steric hindrance related to the growth of the N-acyl chain.

Abbreviations used: NAPE, N-acyl l-α-phosphatidylethanolamine; NAPE-2, N-acetyl 1,2-dihexadecanoyl l-α-phosphatidylethanolamine; NAPE-4, N-butanoyl 1,2-dihexadecanoyl l-α-phosphatidylethanolamine; NAPE-6, N-hexanoyl 1,2-dihexadecanoyl l-α-phosphatidylethanolamine; NAPE-16, N-hexadecanoyl 1,2-dihexadecanoyl l-α-phosphatidylethanolamine; PE, l-α-phosphatidylethanolamine; PLA2, phospholipase A2; PLC, phospholipase C; TFA, trifluoroacetic acid; lyso-N-tBOC-PE, lyso-N-t-butoxycarbonyl-1-hexadecanoyl PE; MC/SD method, Monte Carlo/stochastic dynamics method.

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