Photoaffinity labeling of Crotalus atrox phospholipase A2 by a substrate analogue.

Abstract

A photolabile analogue of phosphatidylethanolamine (photolabile PE analogue), 1,2-di-O-hexylglycero-3-(ethyl diazomalonamidoethyl phosphate), was synthesized in nonisotopic and 14C-radiolabeled form and in both the L configuration (that of the naturally occurring phospholipids) and the racemic form. When the unlabeled racemic compound was photolyzed in the presence of phospholipase A2 of Crotalus atrox, extensive enzyme inactivation was observed. The rate of inactivation was stimulated by Ca2+ and by formation of micelles of the photolabile compound. The dihexyl ether analogue of phosphatidylethanolamine protected the enzyme from inactivation. Phospholipase A2 gave rise to a covalently labeled polypeptide when irradiated in the presence of either L or racemic 14C-labeled photolabile PE analogue. The racemic compound labeled both the N-terminal region (residues 1--15) and the central region (residues 43--97) of the polypeptide while the L compound labeled only the N-terminal region. The lone methionine at position 10 of the C. atrox phospholipase A2 permitted degradation by cyanogen bromide, which showed that labeling by the L compound was restricted to the first ten amino acid residues at the N-terminal end. Phospholipase A2 has an absolute specificity for L-phospholipids, and D-phospholipids are competitive inhibitors. The results of these studies underscore the importance of the head-group region of the phospholipid in phospholipase--substrate interactions and suggest that the two optical isomers of the substrate may be rather differently oriented on the enzyme surface.