Photochemical cross-linking of neighboring residues in protein-nucleic acid complexes: rnase and pyrimidine nucleotide inhibitors.
Iradiation of the stable complexes formed between RNase and its competitive inhibitors cytidine 2'(3'),5'-diphosphate (pCp), and uridine 2'(3'),5'-diphosphate (pUp), resulted in covalent bond formation between the pyrimidine nucleotides and the enzyme. The photochemical reactions were initiated by ultraviolet light of lambda greater than 300 mn, employing acetone as a photosensitizer. This method was found to yield less undesired by-products, particularly photolyzed amino acids and aggregates resulting from protein-to-protein cross-linking, than the direct method of irradiation with light of lambda 260 nm. Tryptic digrestion of the modified protein, and chromatographic analysis of the peptides thus obtained, revealed a single and specific peptide which bacame covalently linked to both nucleotide inhibitors. The amino acid composition of this peptide is consistent with the sequence Asn-67-Arg-85 of RNase. Part of this peptide (residues 78 through 83) is close to the enzyme's binding site for the pyrimidine moiety of the nucleotides. Denatured RNase failed to cross-link to the inhibitors, and the extent of pUp cross-linking could be reduced by the addition of another competitive inhibitor (3'-UMP). Finally, the presence of excess inhibitor in the irradiation mixture did not lead to nonspecific cross-linking. This indicates that specificity is also achieved due to the fact that unbound excited inhibitor molecules do not react with the protein but prefer to follow different and faster reaction paths.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D003597:Cytosine Nucleotides / Q000494:pharmacology / Q000528:radiation effects; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011830:Radiation Effects; D012260:Ribonucleases / Q000378:metabolism; D014466:Ultraviolet Rays; D014500:Uracil Nucleotides / Q000494:pharmacology / Q000528:radiation effects
D000596:Amino Acids; D003597:Cytosine Nucleotides; D010446:Peptide Fragments; D014500:Uracil Nucleotides; D012260:Ribonucleases