High-resolution refinement of orthorhombic bovine pancreatic phospholipaseA(2)

The X-ray structure of recombinant bovine pancreatic phospholipase A(2) (PL A2), which specifically catalyzes the cleavage of the sn-2 acylester bond o f phospholipids, has been refined at 1.5 Angstrom resolution. The crystal b elongs to the space group P2(1)2(1)2(1) with unit-cell parameters a = 47.12 , b = 64.59 and c = 38.14 Angstrom similar to the native enzyme reported pr eviously by Dijkstra et nl. [J. Mel. Biol. (1981), 147, 97-123]. The refine ment converged to an R value of 18.4% (R-free = 22.8%) for 16 374 reflectio ns between 10.0 and 1.5 Angstrom resolution. The surface-loop residues (60- 70) art: ordered in the present orthorhombic recombinant enzyme, but disord ered in the trigonal recombinant enzyme. The active-site residues, His48, A sp99, and the catalytic water superimpose well with the trigonal form. Besi des the catalytic water which is hydrogen bonded to His48, it is often seen that there is a second water attached to the same N atom of His48 and simu ltaneously hydrogen bonded to the O atom of Asp49. It is thought that the s econd water facilitates the tautomerism of His48 for enzyme catalysis, The catalytic water is also hydrogen bonded to the equatorial water coordinated to the calcium ion, In addition to the equatorial water, there is also an axial calcium water and the additional structural water. These five common water molecules are hydrogen bonded to the additional 16 water molecules in the present orthorhombic structure which may further enhance the structura l integrity of the active site. Besides the protein and one calcium ion, a total of 134 water molecules were located in the present high-resolution re finement.