Studies on the role of methionine in porcine pancreatic phospholipase A2.
The unique methionine-15 residue located at the N-terminal site of iso- or beta-phospholipase A2 from porcine pancrease has been specifically carboxymethylated with iodoacetic acid. The modification results in a complete inactivation of the enzymatic activity toward micellar and monomeric substrates. Spectroscopic measurements reveled that the carboxymethylated protein still binds Ca2+ and monomeric substrates with comparable affinities as the native enzyeme. The active site histidine-54 residue in the modified enzyme shows a reactivity toward the active site-directed irreversible inhibitor p-bromophenacylbromide which is identical to that of the native enzyme. The alkylated protein, however, has lost its ability to bind to lipid-water interfaces. Although circular dichroic spectra of the carboxymethylated enzyme display some changes in the tertiary structure as compared with the native enzyme, the alpha-helix content remains rather constant. It is concluded that carboxymethylation of methionine-15 destroys the interface recognition site but has only limited influence on the active site of the molecule. Therefore, it seems that methionine-15 is not involved in the catalytic events but that this residue is part of the interface recognition site which embraces the N-terminal hydrophobic part of the enzyme: Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met.
D000818:Animals; D001665:Binding Sites; D002118:Calcium; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008715:Methionine; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D007461:Iodoacetates; D007527:Isoenzymes; D008715:Methionine; D010740:Phospholipases; D002118:Calcium