A new remote subsite in ribonuclease A.
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(-1) subsite.
Fisher
B M
BM
Departments of Biochemistry and Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Grilley
J E
JE
Raines
R T
RT
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011066:Poly C / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D011066:Poly C; D001120:Arginine; D012259:Ribonuclease, Pancreatic; D008239:Lysine