Jg. Opitz et al., ORIGIN OF THE CATALYTIC ACTIVITY OF BOVINE SEMINAL RIBONUCLEASE AGAINST DOUBLE-STRANDED-RNA, Biochemistry, 37(12), 1998, pp. 4023-4033
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.