Crotalus atrox phospholipase A2. Amino acid sequence and studies on the function of the NH2-terminal region.

Abstract

Many studies indicate a functional role for the N H 2terminal region of phospholipases Az, and this question has been pursued through an analysis of t h e enzyme from Cmtalus atmx venom.Sequence analysis of this enzyme reveals the expected strong homology with the phospholipases Az a and / 3 from the closely related species Crotalus adamanteus.The C. atrox enzyme has 122 residues with seven disulfide bridges.Cleavage with cyanogen bromide at the single methionine (position 10) yields two fragments, an NHz-terminal decapeptide and a 112-residue carboxyl-terminal fragment.Neither the individual purified fragments nor a reconstituted mixture of the two are enzymatically active and the COOH-terminal fragment, which presumably contains much of the native structure of the enzyme, is a monomer in solution, unlike the native enzyme dimer.Akylation of Met-10 by reaction with iodoacetamide at pH 2.6 yields a positively charged sulfonium derivative that is a dimer with specific enzyme activity approximately 150% that of phospholipase toward lecithin and 8.6 times that of phospholipase toward phosphatidic acid.This activation is proposed to result from an electrostatic interaction between the positively charged sulfonium group and the substrate.The N H 2terminal 15-residue peptide forms a stable monolayer at the air-water interface with a collapse pressure of 15 dynes/cm.The area occupied per amino acid residue at the interface is 23.6 A2 indicating that the peptide assumes a relatively compact (presumably an amphipathic, a-helical) conformation.The peptide binds t o single bilayer lecithin vesicles; the dissociation constant is 6.0 X k 1.4 X lo-' M and there are 25.4 2 1.6 peptides bound per vesicle at saturation.These findings, taken as a whole, suggest that the NHz-terminal region of phospholipases Az plays an important mechanistic role in catalysis and that it constitutes a surface-active component of the catalytic site.It may, as well, be of central importance in maintaining the stability of phospholipase dimers, both in solution and at the interface.Phospholipases An (EC 3.1.1.4)catalyze specifically the calcium-dependent hydrolysis of fatty acyl bonds at position 2 of 3-sn-phosphoglycerides.Mammalian pancreas and animal venoms are particularly rich in phospholipase Al and, accord-