Hints on the evolutionary design of a dimeric RNase with special bioactions.
Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed 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) resistance to selective cleavage by subtilisin; and (4) antitumor activity. 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 exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.
Dipartimento di Chimica Organica e Biologica, Università di Napoli Federico II, Naples, Italy.
A
A
Di Donato
V
V
Cafaro
I
I
Romeo
G
G
D'Alessio
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D005075:Biological Evolution; D046911:Macromolecular Substances; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D013381:Subtilisins / Q000737:chemistry
D000970:Antineoplastic Agents; D046911:Macromolecular Substances; D015335:Molecular Probes; D012260:Ribonucleases; D013381:Subtilisins