Asymmetric short-chain phosphatidylcholines: defining chain binding constraints in phospholipases

Abstract

Several short-chain asymmetric lecithins with a total of 14 carbons in the acyl chains (ranging from 1-lauroyl-2-acetylphosphatidylcholine to 1-hexanoyl-2-octanoylphosphatidylcholine) have been synthesized and characterized. The specific activities of phospholipase A2 from cobra venom, phospholipase A2 from porcine pancreas, and phospholipase C from Bacillus cereus toward these lecithins as micelles have been determined. The results of these kinetic studies allow the definition of hydrophobic binding requirements in the active sites of these water-soluble phospholipases. For phospholipase C, with the exception of monomyristoylphosphatidylcholine, each of the asymmetric short-chain lecithins exhibits high activity, comparable to the 14-carbon symmetric short-chain species, diheptanoylphosphatidylcholine. Therefore, for phospholipase C, in addition to the acyl linkages, only a certain degree of hydrophobicity in the fatty acyl chains is requisite for substrate binding and appreciable hydrolysis; there is no chain specificity. The activity of phospholipase A2 from cobra venom toward the same asymmetric lecithins is quite different. As the sn-2 chain lengthens, activity is increased to a maximum for diheptanoyl-PC. Further increase in the number of carbons in the sn-2 chain has no effect on hydrolysis rates. For this enzyme, seven carbons in the sn-2 chain are necessary for optimal activity. In contrast, porcine pancreatic phospholipase A2 activity shows very little dependence on sn-2 chain length.

Mapping the catalytic pocket of phospholipases A2 and C using a novel set of phosphatidylcholines

“…In contrast, PLC behaves as a less discriminating enzyme than PLA # towards this series of substrates ( Figure 2B), as has been extensively documented for other PCs [9][10][11]. However, the magnitude of the differences in catalytic efficiency seen for BHPCs are among the largest ever reported, a likely consequence of the major structural modifications introduced here.…”

“…above the critical micelle concentration [8,9]. Several studies have defined the structural requirements for phospholipids to become substrates of phospholipases [9][10][11]. For optimal PLA # activity, a minimal fatty acyl chain of seven carbon atoms is needed ; no steric hindrance should exist near the glycerol backbone.…”

Discrimination between the regioisomeric 1,2- and 1,3-diacylglycerophosphocholines by phospholipases

“…5A and B, Table 5), which reveal only marginal differences between 1,2-and 1,3-PCs. A certain non-specificity of this enzyme was also previously reported with respect to other substrate analogs such as short-chain lysophosphatidylcholines (El-Sayed et al, 1985) or asymmetric short-chain phosphatidylcholines (Lewis et al, 1990). The slight decrease of the maximum rate with increasing chain length of the acyl moieties in 1,3-PCs is similar to that observed in 1,2-PCs with acyl chain lengths of C 10 -C 16 (Table 1).…”

Carbonothioate Phospholipids as Substrate for a Spectrophotometric Assay of Phospholipase A2

“…The CMC of HPPC was determined using the 31 P NMR method (25,26). HPPC has a CMC value of 0.91 Ϯ 0.03 mM, which is comparable to that of diheptanoyl PC (1.5 Ϯ 0.1 mM) (29). They are structurally very similar also.…”