The hydrolysis of phosphatidyl-alcohols by phospholipases A2: effect of head group size and polarity.
The ability of a variety of secretory phospholipases A2 (sPLA2: EC 3.1.1.4) to bind to and hydrolyse a series of phosphatidyl-alcohol substrates, in the absence of detergent, was explored by both fluorescence-based kinetic and interfacial binding assays. The enzymes used were sPLA2 from porcine pancreas, Naja naja venom and a recombinant human non-pancreatic enzyme. Four dioleoyl phosphatidyl-alcohols were used with different headgroups, methanol, ethanol, propanol and butanol. Comparative kinetic analyses with dioleoyl phosphatidyl-choline, dioleoyl phosphatidyl-glycerol and wheat germ phosphatidyl-inositol are also described. With the phosphatidyl-alcohol series, as the headgroup acyl-chain length increased the susceptibility to hydrolysis decreased. This effect was much more pronounced with the human non-pancreatic and the Naja naja venom enzymes than with the pancreatic enzyme. Maximum activity in this assay system was observed with porcine pancreatic sPLA2 and dioleoyl phosphatidyl-methanol (1440 +/- 167 micromol/min/mg). We demonstrate that the slow rate of hydrolysis of dioleoyl phosphatidyl-propanol by the human non-pancreatic secretory enzyme (4.56 +/- 0.90 micromol/min/mg) is not due to a lack of interfacial binding. The hydrolysis of mixtures of dioleoyl phosphatidyl-choline and dioleoyl phosphatidyl-propanol in various molar proportions by Naja naja sPLA2 suggests good mixing of the two phospholipids with minimal phospholipid domain formation under these assay conditions. We present strong evidence for a stimulation of hydrolysis of phosphatidyl-choline by human non-pancreatic sPLA2 in the presence of as little as 1 mol% phosphatidyl-methanol (<40 fold total rate enhancement). Overall, the results demonstrate that the rates of hydrolysis of anionic phospholipids by sPLA2 vary considerably with the different enzymes from this close structurally related family. The tight binding of the human enzyme to poorly hydrolysable anionic phospholipid vesicles provides a novel mechanism of enzyme inhibition by interfacial sequestration.
Kinkaid
A R
AR
Department of Biochemistry, University of Southampton, UK.
Wilton
D C
DC
D000438:Alcohols / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010741:Phospholipases A / Q000502:physiology*; D010743:Phospholipids / Q000378:metabolism*
D000438:Alcohols; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010743:Phospholipids; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D010741:Phospholipases A; C017251:1,2-oleoylphosphatidylcholine