Chemical Modification of Tyrosine Residues in Ribonuclease T1 with N-Acetylimidazole and p-Diazobenzenesulfonic Acid1

Abstract

The reactions of ribonuclease T1 [EC 3.1.4.8] with N-acetylimidazole and p-diazobenzenesulfonic acid have been investigated to obtain some information on the states and roles of tyrosine residues in the enzyme. Three to four tyrosine residues in the enzyme were acetylated with N-acetylimidazole fairly readily at pH 7.5 without extensive loss of activity toward RNA. Of these, two residues appeared to be acetylated most easily. In the presence of phosphate anion, however, the reactivity of N-acetylimidazole was significantly lowered. Under the reaction conditions used, no acetylation took place in 0.2 m phosphate buffer. On the other hand, one to two tyrosine residues were modified with p-diazobenzenesulfonic acid in 0.2 m phosphate buffer, pH 7.0, without much loss of activity. In 8 M urea all the tyrosine residues were acetylated with nearly complete loss of activity. These results indicated that at least two tyrosine residues are fully exposed while most of the remaining seven tyrosine residues are buried inside the molecule. Judged from the change in CD spectrum, some conformational change appeared to have already occurred when two tyrosine residues were acetylated. Upon removal of all the acetyl groups introduced by incubation with hydroxylamine, the fully acetylated, inactive enzyme regained full activity, although the original native structure did not appear to be completely regenerated from CD spectrum measurements. These results indicated that several of the tyrosine residues in ribonuclease T1 are important in the formation and maintenance of the enzymatically active conformation although they are presumably not directly involved in the active site.