HINTS ON THE EVOLUTIONARY DESIGN OF A DIMERIC RNASE WITH SPECIAL BIOACTIONS

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Caf aro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, V itagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal dom ain swapping of seminal RNase. In an attempt to have a clearer underst anding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was const ructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) res istance to selective cleavage by subtilisin; and (4) antitumor activit y. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the ex change of N-teminal domains; (3) the exchange is the molecular basis f or the RNase antitumor action; and (4) this exchange is not a prerequi site in an evolutionary mechanism for the generation of dimeric RNases .