The structure and function of ribonuclease T1. XXIII. Inactivation of ribonuclease T1 by reversible blocking of amino groups with cis-aconitic anhydride and related dicarboxylic acid anhydrides.
Ribonuclease T1 [EC 3.1.4.8] was inactivated rapidly by treatment at pH 8.0 and 0 degrees C with cis-aconitic anhydride and related dicabroxylic acid anhydrides, including citraconic, maleic, and succinic anhydrides. Under reaction conditions used, roughly 90% inactivation occurred within 30 min. Analyses of the inactivated enzymes indicated that the reaction took place fairly specifically at the alpha-amino group of the N-terminal alanine and the epsilon-amino group of lysine-41. Upon incubation of these inactivated enzymes at pH 3.6 and 37 degreeC, the activity was regenerated to various extents, depending on the nature of the introduced acyl groups. Under these conditions, the enzyme modified with cis-aconitc anhydride or citraconic anhydride recovered much of the origninal activity after 48 h whereas the enzyme modified with maleic anhydride recovered its activity only partially. Practically no activity was regenerated in the case of the enzyme modified with succinic anhydride under these conditions. The inactivation appears to be due mainly to the effect of the carboxyl group introduced at the epsilon-amino group of lysine-41. The results suggest the usefulness of cis-aconitic anhydride as a reversible blocking reagent for amino groups in proteins.
D000156:Aconitic Acid / Q000494:pharmacology; D000812:Anhydrides / Q000494:pharmacology; D001667:Binding, Competitive; D003998:Dicarboxylic Acids / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D011485:Protein Binding; D006163:Ribonuclease T1 / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D000812:Anhydrides; D003998:Dicarboxylic Acids; D004140:Dinitrophenols; D000156:Aconitic Acid; D012260:Ribonucleases; D006163:Ribonuclease T1; D006918:Hydroxyurea