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J Biol Chem, Vol. 273, Issue 51, 34134-34138, December 18, 1998
From the Departments of Biochemistry and Chemistry, University of
Wisconsin The interaction between bovine pancreatic
ribonuclease A (RNase A) and its RNA substrate extends beyond the
scissile bond. Enzymic subsites interact with the bases and the
phosphoryl groups of a bound substrate. We evaluated the four cationic
residues closest to known subsites for their abilities to interact with a bound nucleic acid. Lys-37, Arg-39, Arg-85, and Lys-104 were replaced
individually by an alanine residue, and the resulting enzymes were
assayed as catalysts of poly(cytidylic acid) (poly(C)) cleavage. The
values of Km and
kcat/Km for poly(C) cleavage were affected only by replacing Arg-85. Moreover, the contribution of Arg-85 to the binding of the ground state and the
transition state was uniform---Km increased by
15-fold and kcat/Km
decreased by 10-fold. The contribution of Arg-85 to binding was also
apparent in the values of Kd for complexes with
oligonucleotides of different length. This contribution was dependent
on salt concentration, as expected from a coulombic interaction between
a cationic side chain and an anionic phosphoryl group. Together, these
data indicate that Arg-85 interacts with a particular phosphoryl group
of a bound nucleic acid. We propose that Arg-85 comprises a new distal
subsite in RNase A---the P(
Madison, Madison, Wisconsin 53706
1) subsite.
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