COMPARATIVE STUDIES OF TYROSINE MODIFICATION IN PANCREATIC PHOSPHOLIPASES .2. PROPERTIES OF THE NITROTYROSYL, AMINOTYROSYL, AND DANSYLAMINOTYROSYL DERIVATIVES OF PIG, HORSE, AND OX PHOSPHOLIPASES-A2 AND THEIR ZYMOGENS

Specifically nitrated phospholipases A2 and the corresponding zymogens from pig, horse, and ox pancreas have been used to investigate the role of particular tyrosine residues in phospholipid and Ca2+ binding. Ultraviolet difference spectroscopy allowed direct binding studies with n-alkyl-phosphocholines as the substrate analogues. After reduction and dansylation of the nitrated enzymes, the interaction with phospholipids could also be monitored by fluorescence spectroscopy. Due to the considerable enzymatic activity of the nitrotyrosine, aminotyrosine, and dansylaminotyrosine phospholipases, their action on monomolecular surface layers of l, 2-didecanoyl-sn-glycero-3-phosphocholine as substrate was investigated. From this study it is concluded that the invariant tyrosine-69 in horse, pig, and ox phospholipases A2 and the variable tyrosine-19 in the horse enzyme are part of the previously proposed interface recognition site [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A., & de Haas, G. H. (1975) Biochemistry 14, 5387-5394] and that tyrosine-123 in the porcine enzyme is not involved in binding to lipid-water interfaces. In contrast to the modified phospholipases A2, the corresponding nitrotyrosine, aminotyrosine, and dansylaminotyrosine zymogens do not bind to micellar substrate analogues. Monomer phospholipid binding (as monitored by ultraviolet difference spectroscopy) induces a strong hydrophobic perturbation of nitrotyrosine-69 and -19, and again the microenvironment of nitrotyrosine-123 is not changed. Because of the lower pK of nitrotyrosine as compared to tyrosine, measurements were also performed at pH 8.0 where nitrotyrosine is negatively charged. Both monomer binding and penetrating capacity of nitrotyrosine-69 phos-pholipase are severely distorted, probably because of charge repulsion between the negatively charged nitrotyrosine and the phosphate moiety of the phospholipid. Finally, it could be shown by ultraviolet difference spectroscopy that binding of the essential cofactor Ca2+ to the tyrosine-69 nitrated phospholipases results in a conformational change influencing the microenvironment of tyrosine-69. Horse nitrotyrosine-19 phospholipase and pig nitrotyrosine-123 phospholipase are not perturbed by Ca2+ binding. These results are in agreement with the recently proposed three-dimensional structure of the bovine phospholipase A2 [Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaelen, P. J. (1978) J. Mol. Biol. 124, 53-60]. © 1979, American Chemical Society. All rights reserved.