CRYSTAL STRUCTURE OF THE 29KDa SUBUNIT OF THE RECOMBINANT PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE. Y. S. Ho^{[[paragraph]]}, Z. Dauter^[[yen]], Keizo Inoue^+, Z. S. Derewenda^{[[paragraph]], [[paragraph]]}Department of Molecular Physiology & Biological Physics, University of Virginia, ^[[yen]]EMBL-Hamburg, Germany, ⁺Department of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo.

Platelet-activating factor(PAF) is a potent phospholipid mediator involved in many physiological events. The enzyme that is responsible for breaking down PAF exists both intracellularly in cells and extracellularly in the blood plasma. PAF-acetylhydrolase(PAF-AH) hydrolyses the acetyl moiety of PAF at the sn-2 position. PAF-AH differs from other phospholipase A2(PLA2) in three aspects. First, it does not require Ca²⁺ ions for its activation. Second, it differs from all known PLA2 in that it is serine dependent, however, it differs from other serine hydrolases in that it does not have the consensus GXSXG sequence Third, unlike PLA2 which cleave the ester bond at the sn-2 position of phospholipids regardless of it length, PAF-AH has a specificity for acyl moiety not longer than C4. The intracellular form has been isolated from bovine brain, the protein is a heterotrimeric enzyme consisting of a 29- 30- and 45kDa subunits. The 29kDa subunit has been successfully isolated and crystallized.

The structure was solved by MIR and optimized anomalous scattering methods based on three derivatives at 2.2Å resolution. The model has an R-factor of 24.5% and refinement process using XPLOR is underway. The general fold of the enzyme belongs to the [[alpha]]/[[beta]] family with five parallel [[beta]]-sheets in the center and the [[alpha]] helices packed on both sides of the [[beta]]-sheets. It has a unique fold that is different from all other [[alpha]]/[[beta]] hydrolases and it is the first characterized PLA2 that is serine dependent. Gel filtration column chromatography of the enzyme showed that it forms a homodimer. From the model, the active site is found to be buried at the interface between the homodimer. The active site residues consist of S47, D192 and H195. Experiments are underway to compare the differences between PAH-AH and PLA2 regarding the specificity of PAF-AH for short chain acetyl moiety. The model will be refined further to 1.7Å using, data collected from beamline BW7B at DESY by cryogenic method.