Role of the interfacial binding domain in the oxidative susceptibility of lecithin:cholesterol acyltransferase

We had previously shown that the Cholesterol esterification activity of lecithin:cholesterol Acyltransferase (LCAT) is destroyed by oxidation, but still it retains the ability to hydrolyse water-soluble substrates. This suggested that the inactivation of the Enzyme is not due to its catalytic function, but due to a loss of its hydrophobic binding. Since recent studies have shown that a tryptophan residue in the putative interfacial domain (Trp(61)) is critical for the activity, we determined the possible role of this residue in the oxidative susceptibility and substrate specificity of LCAT by site-directed mutagenesis. Deletion of Trp(61) resulted in a 56% loss of Cholesterol esterification (LCAT) activity, but the Phospholipase A(2) (PLA(2)) and the esterase activities of the Enzyme were stimulated slightly. Replacing Trp(61) with another aromatic residue [Trp(61)-->Tyr (W61Y)] resulted in an increase in all activities (14-157%), whereas replacing it with an aliphatic residue [Trp(61)-->Gly (W61G)] caused a dramatic loss of LCAT (-90%) and PLA(2) (-82%) activities, but not the esterase activity (-5%). W61Y was the most sensitive to oxidation, whereas W61G was the most resistant, with respect to the LCAT and PLA(2) activities. However, the activities which do not involve interfacial binding, namely the esterase activity and the transesterification of short-chain Phospholipids, were more resistant to oxidation in all LCATs, indicating a selective loss of the interfacial binding by oxidation. Furthermore, replacing the two cysteines (Cys(31) and Cys(184)) in the Trp(61) deletion mutant caused additional resistance of the Enzyme to oxidizing agents, showing that both domains of the Enzyme contribute independently to its oxidative susceptibility. Since the hydrolysis of truncated Phospholipids, generated during the oxidation of low-density lipoproteins, does not require the interfacial-binding domain, our results suggest that LCAT may take part in the detoxification of these compounds even after the loss of its Cholesterol esterification function.