ORIGIN OF THE CATALYTIC ACTIVITY OF BOVINE SEMINAL RIBONUCLEASE AGAINST DOUBLE-STRANDED-RNA

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, t he well-known RNase A. Reported here are site-directed mutagenesis exp eriments that identify the sequence determinants of this enhanced cata lytic activity. These experiments have been guided in part by experime ntal reconstructions of ancestral RNases from extinct organisms that w ere intermediates in the evolution of the RNase superfamily. It is sho wn that the enhanced interactions between bovine seminal RNase and dou ble-stranded nucleic acid do not arise from the increased number of ba sic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at pos itions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A stru ctural model is presented to explain these data, the use of evolutiona ry reconstructions to guide protein engineering experiments is discuss ed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.