The role of lysine in the action of bovine pancreatic ribonuclease A.
The involvement of lysine residues in the active site of pancreatic ribonuclease has been investigated by assessing (a) the degree of substrate and substrate analogue protection of individual lysine residues against acetylation, and (b) the individual contribution of remaining unacetylated lysine residues to the total catalytic activity of the enzyme. Different substrate analogues (RNA digest, CMP, ATP, and pyrophosphate) were found to give different degrees of protection against acetylation with acetic anhydride. Instead of the expected specific protection of active site lysine residues such as lysine-7 and lysine-41, however, a general decrease in reactivity of all the lysines was observed when the substrate analogues were present during the acetylation. The fraction of enzymatic activity remaining in the protected samples was consistently greater than the fraction of any one lysine remaining unacetylated, and was found to correspond fairly well with the sum of the fractions of unacetylated lysine-7, lysine-41, and a third residue, tentatively assigned as lysine-66. This is consistent with other observations of ribonuclease which suggest that while no lysine residue interacts with substrate and substrate analogues in the formation of the Michaelis-Menten complex, a lysine amino group is required for catalysis. It is proposed that this lysine amino group can be supplied by any one of two or three lysine residues (7, 41, and 66) located close to the substrate binding site.
D000085:Acetates; D000596:Amino Acids / Q000032:analysis; D000812:Anhydrides; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D008239:Lysine / Q000502:physiology; D010179:Pancreas / Q000201:enzymology*; D011485:Protein Binding; D012260:Ribonucleases / Q000502:physiology
D000085:Acetates; D000596:Amino Acids; D000812:Anhydrides; D012260:Ribonucleases; D008239:Lysine