BINDING OF ACTIVE-SITE-DIRECTED LIGANDS TO PHOSPHOLIPASE-A2 - IMPLICATIONS ON THE MOLECULAR CONSTRAINTS AND CATALYTIC MECHANISM

Molecular constraints for the localization of active site directed lig ands (competitive inhibitors and substrates) in the active site of pho spholipase A2 (PLA2) are characterized. Structure activity relationshi ps with known inhibitors suggest that the head : group interactions do minate the selectivity as well as a substantial part of the affinity. The ab initio fitting of the amide ligands in the active site was carr ied out to characterize the head group interactions. Based on a system atic coordinate space search, formamide is docked with known experimen tal constraints such as coordination of the carbonyl group to Ca2+ and hydrogen bond between amide nitrogen and ND1 of His48. An optimal pos ition for a bound water molecule is identified and its significance fo r the catalytic mechanism is postulated. Unlike the traditional ''pseu do-triad'' mechanism, the ''Ca-coordinatedoxyanion'' mechanism propose d here invokes activation of the catalytic water to form the oxyanion in the coordination sphere of calcium. As it attacks the carbonyl carb on of the ester, a near-tetrahedral intermediate is formed. As the sec ond proton of the catalytic water is abstracted by the ester oxygen, i ts reorientation and simultaneous cleavage form hydrogen bond with ND1 of His48. In this mechanism of esterolysis, a catalytic role for the water co-ordinated to Ca2+ is recognised.