Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives

• Published in: Biochimica et biophysica acta Vol. 747; no. 3; pp. 253 - 262
• Main Authors: DE CARO, J. D, BEHNKE, W. D, BONICEL, J. J, DESNUELLE, P. A, ROVERY, M
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 28.09.1983; North-Holland
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Colipases; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Lipase - metabolism; Methane - analogs & derivatives; Micelles; Pancreas - enzymology; Proteins; Swine; Taurodeoxycholic Acid; Tetranitromethane; Tyrosine; Tyrosine; Nitration; Panel; Chemical modification; Structure activity relation; Cofactor; Circular dichroism; Pig
• ISSN: 0006-3002; 1878-2434

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.