Micellar Systems for Defining the Active Site of Phospholipase-A2: Methyl Branching in Short-Chain Lecithins

Abstract

Short-chain lecithin micelles have been used extensively to characterize the activity of water soluble phospholipases. In order to understand the mechanism of such esterases we have synthesized and characterized a series of micellar seven-carbon fatty acyl lecithins with varied acyl chain branching: diheptanoyl phosphatidylcholine (PC), di(2-methyl)hexanoyl-PC, di(3-me thy1)hexanoy1-PC, di(4,4-me thy1)pentanoy1-PC, di(5-methyl)hexanoyl-PC, l-heptanoyl-2-(2-methyl)hexanoyl-PC, and l-(3-methyl)hexanoyl-2-heptanoyl-PC. Compared to diheptanoy1-PC, the lecithins with both chains branched are poor substrates and good inhibitors of phospholipase-A2. When only one acyl chain bears a branch methyl group, the hybrid is a substrate comparable to the linear lecithin. Such variations in phospholipase activity towards these systems might be caused by altered micelle surface properties or altered phospholipid structure and do not reflect binding constraints at the enzyme active site. Analyses of micellar structure and dynamics with H and C NMR spectroscopy, quasielastic light scattering and critical micelle concentrations indicate little significant difference in the conformation and aggregate structure of these branched chain lecithins from the linear compound, even when the methyl group is adjacent to the carbonyl group. The low hydrolytic activity of phospholipase-A2 against the branched chain micellar lecithins can then be attributed to a steric interaction of both bulky fatty acyl chains with the enzyme that prevents a catalytically efficient conformational change in the protein. U.V. difference spectroscopy of the different micellar lipids binding to the enzyme supports this view.