Coulombic effects of remote subsites on the active site of ribonuclease A

The active-site cleft of bovine pancreatic ribonuclease A (RNase A) is line d with cationic residues that interact with a bound nucleic acid. Those res idues interacting with the phosphoryl groups comprise the P0, P1, and P2 su bsites, with the scissile P-O-5' bond residing in the P1 subsite. Coulombic interactions between the P0 and P2 subsites and phosphoryl groups of the s ubstrate were characterized previously [Fisher, B, M., Ha, J.-H., and Raine s, R. T. (1998) Biochemistry 37, 12121-12132]. Here, the interactions betwe en these subsites and the active-site residues His12 and His119 are describ ed in detail. A protein variant in which the cationic residues in these sub sites (Lys66 in the P0 subsite and Lys7 and Arg10 in the P2 subsite) were r eplaced with alanine was crystallized, both free and with bound 3'-uridine monophosphate (3'-UMP). Structures of K7A/R10A/K66A RNase A and the K7A/R10 A/K66A RNase A 3'-UMP complex were determined by X-ray diffraction analysis to resolutions of 2.0 and 2.1 Angstrom, respectively. There is little obse rvable change between these structures and that of wild-type RNase A, eithe r free or with bound 3'-cytidine monophosphate. K7A/R10A/K66A RNase A was e valuated for its ability to cleave UpA, a dinucleotide substrate that does not span the P0 or the P2 subsites. In comparison to the wild-type enzyme, the value of k(cat) was decreased by 5-fold and that of k(cat)/K-m was decr eased 10-fold, suggesting that these remote subsites interact with the acti ve site, These interactions were characterized by determining the pK(a) val ues of His12 and His119 at 0.018 and 0.142 M Na+, both in wild-type RNase A and the K7A/R10A/K66A variant. The side chains of Lys7, Arg10, and Lys66 d epress the pK(a) values of these histidine residues, and this depression is sensitive to the salt concentration. In addition, the P0 and P2 subsites i nfluence the interaction of Hisl2 and His119 with each other, as demonstrat ed by changes in the cooperativity that gives rise to microscopic pK(a) val ues. Finally, the affinity of 3'-UMP for wild-type RNase A and the K7A/R10A /K66A variant at 0.018 and 0.142 M Na+ was determined by isothermal titrati on calorimetry. 3'-UMP binds to the variant protein with 5-fold weaker affi nity at 0.018 M Na+ and 3-fold weaker affinity at 0.142 M Na+ than it binds to wild-type RNase A. Together these data demonstrate that long-range Coul ombic interactions are an important feature in catalysis by RNase A.