Formation of an RNase A derivative containing an aminosuccinyl residue in place of asparagine 67

At acidic pH, Asp67 and beta-Asp67 (beta-Asp: isoaspartic acid residue) derivatives of RNase A, obtained by selective deamidation of the parent enzyme, spontaneously produces a new derivative containing an aminosuccinyl residue (Asu). The overall secondary structure of the protein chain does not change as a consequence of this substitution, while the catalytic activity on RNA is reduced to about 25%. The pH dependence of the first-order rate constants for the Asu formation has a bell-shaped profile, the maximum being close to the pK(a) of the aspartic acid side chains. Moreover, the values of the rate constants are of the same magnitude of those measured for Asp-containing peptides whose sequence mimics the Asu formation site of the enzyme. This feature indicates that Asp67 and beta-Asp67 residues in the deamidated RNase A derivatives are sited in a region flexible enough to permit the cyclization of the carboxylic side chain to succinimide ring. These results are discussed at the light on to the three-dimensional structure and the thermodynamic stability of the aspartic acid derivatives of RNase A.