Purification and characterization of a mutant human platelet phospholipase A 2 expressed in Escherichia coli

Both methionine residues in phospholipase A 2 (PLA 2 ) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine‐less mutant has been expressed as a Cro‐LacZ fusion protein in Escherichia coli , from which a pro‐PLA 2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A 2 (HP‐PLA 2 ) by a leucine residue, and the introduction of a methionine at a position just preceding the HP‐PLA 2 sequence. This protein was expressed in E. coli as a 68‐kDa Cro‐LacZ fusion protein. CNBr cleavage liberated the HP‐PLA 2 fragment which was reoxidized in vitro. The [Met8→Leu]HP‐PLA 2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p ‐Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2‐dioctanoyl‐ sn ‐glycero‐3‐phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2‐dioctanoyl‐ sn ‐glycero‐3‐phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol > phosphatidylethanolamine ≫ phosphatidylcholine. The enzyme has low activity on monomeric 1,2‐diheptanoyl‐ sn ‐glycero‐3‐phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP‐PLA 2 , porcine pancreatic PLA 2 and PLA 2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n ‐hexadecylphosphocholine. The HP‐PLA 2 has a high apparent K d (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA 2 . In mixed micelles of taurodeoxycholate and 1,2‐didodecanoyl‐ sn ‐glycero‐3‐phosphocholine, the competitive inhibition of HP‐PLA 2 by the R and S enantiomers of 2‐tetradecanoylaminohexanol‐1‐phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine ≪ phosphoethanolamine < phosphoglycol. The best inhibitor, ( R )‐2‐tetradecanoylaminohexanol‐1‐phosphoglycol, binds 2200 times stronger than the substrate to the HP‐PLA 2 active site.