Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism.
It is known that His-48 is part of the active center in pancreatic phospholipase. To further elucidate the role of histidine-48 in the active center of pancreatic phospholipase A2, we have modified the enzyme with a number of bromo ketones and methyl benzenesulfonates. Rapid methylation occurred with methyl p-nitrobenzenesulfonate. Methylated phospholipase shows total loss of enzymatic activity whereas binding of substrate and the cofactor Ca2+ remains intact. Amino acid analysis of methylated equine phospholipase showed the loss of the single molecule of histidine and the formation of one molecule of 2-amino-3-(1-methyl-5-imidazolyl)propanoic acid (1-methylhistidine). Equine phospholipase was also modified by [13C]methyl p-nitrobenzenesulfonate and the methylated enzyme was studied by 13C NMR. The results indicate that the proton on the nitrogen in position 3 of the imidazole ring is involved in a strong interaction with a buried carboxylate group, thereby hindering rotation of the imidazole ring, and that the nitrogen in position 1 is involved in catalysis. These data are in full agreement with the three-dimensional structure at 1.7-A resolution of bovine pancreatic phospholipase. A catalytic mechanism is proposed in which a water molecule which is close to the nitrogen at position 1 of the imidazole ring of the Asp-99-His-48 couple acts as the nucleophile. A comparison is made between phospholipase A2 and the serine esterases.
D000818:Animals; D001557:Benzenesulfonates / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D006639:Histidine; D006736:Horses; D007659:Ketones / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013552:Swine
D001557:Benzenesulfonates; D007659:Ketones; D006639:Histidine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium