Crystal structures of the free and anisic acid bound triple mutant of phospholipase A2.

Phospholipase A2 catalyses the hydrolysis of the ester bond of 3-sn-phosphoglycerides. Here, we report the crystal structures of the free and anisic acid-bound triple mutant (K53,56,120M) of bovine pancreatic phospholipase A2. In the bound triple mutant structure, the small organic molecule p-anisic acid is found in the active site, and one of the carboxylate oxygen atoms is coordinated to the functionally important primary calcium ion. The other carboxylate oxygen atom is hydrogen bonded to the phenolic hydroxyl group of Tyr69. In addition, the bound anisic acid molecule replaces one of the functionally important water molecules in the active site. The residues 60-70, which are in a loop (surface loop), are disordered in most of the bovine pancreatic phospholipase A2 structures. It is interesting to note that these residues are ordered in the bound triple mutant structure but are disordered in the free triple mutant structure. The organic crystallization ingredient 2-methyl-2,4-pentanediol is found near the active site of the free triple mutant structure. The overall tertiary folding and stereochemical parameters for the final models of the free and anisic acid-bound triple mutant are virtually identical.