STRUCTURE AND FUNCTION OF THE CATALYTIC SITE MUTANT ASP-99 ASN OF PHOSPHOLIPASE-A(2) - ABSENCE OF THE CONSERVED STRUCTURAL WATER

To probe the role of the Asp-99...His-48 pair in phospholipase A(2) (P LA2) catalysis, the X-ray structure and kinetic characterization of th e mutant Asp-99 --> Asn-99 (D99N) of bovine pancreatic PLA2 was undert aken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemi stry 30:11801-11811). The 1.9-Angstrom X-ray structure of the mutant s howed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mut ant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrog en bonded to His-57 resulting in the inactive tautomeric form and henc e the loss of enzymatic activity. Although the geometry of the catalyt ic triad in the PLA2 mutant remains the same as in the WT, we were sur prised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.