Modification of amino acids and bovine pancreatic ribonuclease A by kethoxal.
Kethoxal (3-ethoxy-2-ketobutanal) reacts with the guanidino group of Nalpha-acetylarginine to produce four derivatives, reactive to periodate, stable at pH 7, with 15% reverting to arginine on acid hydrolysis. Other amino acids with blocked alpha-amino groups do not react, except the epsilon-amino of lysine (slowly). The pK of the mixed Kethoxal-Nalpha-acetylarginine derivatives is 5.8-6.1. Kethoxal reacts at neutral pH with arginyl residues of bovine pancreatic ribonuclease A. In the presence of an active-site ligand, arginine-39 and arginine-85 react at about equal rates. The loss of enzymic activity at pH 7 is proportional to the combined loss of these residues. The enzymic activity toward RNA is 20-25% of that of native RNAase at pH 7, and 90-100% at pH 5. In the absence of an active site ligand, arginine-10 is also modified with the loss of almost all enzymic activity, although arginine-10 is not an active-site residue. Arginine-33 is unreactive. Kethoxal-modified RNAase undergoes cross-linking in solution at pH 7 or in the freeze-dried state, Incubation at pH 9 in the presence of homoarginine results in partial regeneration of arginyl residues and activity at pH 7. Kethoxal modification of arginines-39 and -85 appears to raise the pK of lysine-41 by about 1 unit, as indicated ty the pH dependence of arylation by 2-carboxy-4,6-dinitrochlorobenzene. The claims of Patthy and Smith (J. Biol, Chem. (1975) 250, 565-569), and of Takahashi (J. Biol. Chem. (1968) 243, 6171-6179) that arginine-39 is a more important functional residue than is arginine-85 are questioned.
D000447:Aldehydes; D000596:Amino Acids; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives; D001665:Binding Sites; D002074:Butanones; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides; D011485:Protein Binding; D012260:Ribonucleases / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D000447:Aldehydes; D000596:Amino Acids; D002074:Butanones; D010446:Peptide Fragments; D010455:Peptides; D001120:Arginine; C005135:kethoxal; D012260:Ribonucleases; D014357:Trypsin; D008239:Lysine