We have recently shown that an endogenous phospholipase A2 from bovine erythrocytes does not hydrolyse NAPEs (N-acyl l-?-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-?-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.
Keywords
N-acyl phosphatidyl ethanolamine,
molecular dynamics,
phospholipase A2