CATALYTIC MECHANISM OF PHOSPHOLIPASE A2

Abstract

Phospholipases A2 (MW 14,000) specifically hydrolyse the 2-acyl linkage of phosphoglycerides in a calcium dependent reaction. From the three-dimensional structure of native bovine pancreatic phospholipase Az at 1.7 A resolution in conjunction with biochemical evidence the architecture of the active site became apparent and a mechanism for the hydrolysis of both monomeric as well as aggregated phospholipids could be proposed. In this mechanism a water molecule activated by His48 was suggested as the nucleophile. The enzyme possesses an extended area around the entrance to the active site that is involved in the binding of aggregated substrates. Pro-phospholipase can not degrade these aggregated substrates and it appears that in pro-phospholipase part of this binding area is flexible. The three-dimensional structure of a phospholipase Az mutant complexed with a substrate analogue shows that the substrate analogue is bound to the calcium ion in the enzyme’s active site both by its phosphate group and by the carbonyl oxygen of the fatty acid residue bound at the C2 atom of the glycerol backbone. The sn-2 chain of the inhibitor makes extensive hydrophobic contacts with the disulfide bond between residues 29 and 45, and the side chains of residues Leu2, Phe5, lle9, Leu19, Phe22 and Tyr52. The hydroxyl group of Tyr69 is hydrogenbonded to one of the oxygen atoms of the inhibitor’s phosphate group.