Probing the role of C‐1 ester group in Naja naja phospholipase 
A₂–phospholipid interactions using butanetriol‐containing 
phosphatidylcholine analogues

Abstract

To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A 2 ±glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A 2 . Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A 2 ± phospholipid interactions.Keywords: phospholipase A 2 ; phosphatidylcholine; phospholipid conformation; butanetriol analogue.Cobra venom phospholipase A 2 is a Ca 2+ -dependent enzyme that catalyses the hydrolysis of the fatty acyl group at the sn-2 position of membrane phospholipids. It is composed of a single polypeptide chain of 119 amino acids which contains seven disulphide bonds [1]. The crystal structures of phospholipase A 2 from several sources have been determined [1±3], and the amino acids that form the enzyme catalytic site have been reported to be highly conserved [1]. Also, the structure of the catalytic surface has been shown to be exactly identical regardless of the degree of evolutionary divergence, state of oligomerization, ionic strength, or pH of the crystallization conditions [3,4].Binding of phospholipase A 2 with phosphatidylcholines involves interactions of the enzyme active site calcium with the phospholipid phosphate oxygen and the sn-2 carbonyl oxygen followed by the hydrophobic interactions between the phospholipid fatty acyl chains and the enzyme catalytic site [2±5]. Apart from the sn-2 acyl chain [5], the acyl chain at the sn-1 position also plays an important role in phospholipase A 2 ±glycerophos-pholipid interactions [6,7]. It has been reported that an increase in the hydrophobicity of the sn-1 acyl residue increases the affinity between the phospholipid molecule and the enzyme [6]. However, it is not yet known whether the ester moiety (-CO-O-) of the sn-1 acyl chain plays any role in phospholipase A 2 ± glycerophospholipid interactions. To investigate this we introduced one additional methylene residue between the glycerol C1 and C2 carbon atoms and then studied the interactions of the resulting modified phospholipids (Fig. 1) with Naja naja phospholipase A 2 . Results of these studies indicate that the sn-1 ester moiety plays an important role in binding of the enzyme with the substrate. MATERIALS AND METHODS MaterialsPhospholipase A 2 from N. naja snake venom, sn-glycero-3-phosphocholine, 6-N-(7-nitrobe...