A New Remote Subsite in Ribonuclease A

Abstract

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 K m and k cat /K m 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 uniformOK m increased by 15-fold and k cat /K m decreased by 10-fold. The contribution of Arg-85 to binding was also apparent in the values of K d 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 AOthe P(؊1) subsite.The efficiency of enzymatic catalysts is a source of continued interest and inspiration as molecular scientists strive to design new catalysts. A distinguishing characteristic of enzymic catalysts is that they bind to their substrates (1, 2). Binding energy is necessary to compensate for the loss of translational and rotational entropy and for any destabilization of the substrate required to reach the transition state (3, 4). Multivalent contacts between an enzyme and substrate provide much of this required binding energy. Indeed, many enzymes that cleave polymeric substrates have subsites that interact with monomeric units of the substrate.Bovine pancreatic ribonuclease A (RNase A; 1 EC 3.1.27.5) is a classic model for revealing the physical, chemical, and biological properties of enzymes (5, 6). RNase A is a 13.7-kDa endoribonuclease that binds RNA in a cationic cleft and cleaves on the 3Ј-side of pyrimidine residues. The cleft contains subsites (B1, B2, and B3) that interact specifically with bases and subsites (P0, P1, and P2) that interact with phosphoryl groups (7,8). The specificity of RNase A for pyrimidine bases is because of exclusion of the larger purine bases from the B1 subsite (9). The B2 and B3 subsites prefer to bind purine bases. His-12, His-119, and Lys-41 of the P1 subsite are the residues most central to the catalytic function of the enzyme. The amino acid residues that comprise the P0 (Lys-66) and P2 (Lys-7 and Arg-10) subsites increase the affinity with which the substrate binds to the enzyme and participate indirectly in catalysis (10 -12).Some data portend the existence of additional RNase A binding sites beyond those characterized previously. Three-dimensional structures derived from x-ray diffraction analyses reveal a line of cationic res...

Ferrocenylnaphthalene Diimide-based Electrochemical Ribonuclease Assay

“…The RNase A detection method previously reported [3][4][5][6]26 was 0.5 pg RNase A and the the detection limit of this method is in good agreement with that previously reported. These results showed that RNase could be detected by the mRNA-immobilized electrode coupled with 1.…”

“…[20][21][22] The immobilization of mRNA on the electrode thus obtained was then conducted based on the fact that deoxyguanosine or deoxycytidine of DNA could react with NHS-activated carboxylic acid on the electrode. 20,21 Since the mRNA used in this experiment had a lot of guanosine and cytidine, 24 the mRNA was expected to be immobilized through a similar reaction. Poly(A) + RNA from mouse kidney as mRNA had an averaged length of 1 -2 kb, confirmed by an Agilent 2100 bioanalyzer (data not shown).…”

“…[3][4][5][6]26 This electrochemical detection method, based on decreasing current still remains to be improved. However, we could succeed in a highly sensitive electrochemical detection of RNase A, even under the conditions employed here.…”

“…So far, a hypersensitive fluorometric RNase detection method is known with the RNase-substrate oligonucleotide having fluorescein and rhodamine dyes at both termini. [3][4][5][6] This oligonucleotide substrate shows fluorescence quenching of the fluorescein by the proximal rhodamine in an aqueous solution. However, in the presence of RNase, the fluorescence of the fluorescein was recovered by cleavage of the oligonucleotide due to RNase digestion.…”

Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase

“…Indeed, binding interactions between enzymes and substrates, both directly at the site of chemical transformation and with nonreacting portions of the substrate have been shown to contribute to catalysis (e.g., refs. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. The mechanisms by which these noncovalent interactions can assist catalysis have been widely discussed, as briefly described in the following two paragraphs.…”