A RESIDUE TO RESIDUE HYDROGEN-BOND MEDIATES THE NUCLEOTIDE SPECIFICITY OF RIBONUCLEASE-A

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of t he P-O-5' bond of RNA after residues bound in the enzyme's B1 subsite. This subsite binds to cytidine 30-fold more tightly than to uridine a nd >10(5)-fold more tightly than to adenine. Structural studies had su ggested that the hydroxyl group of Thr45 can interact directly with th e base of a bound nucleotide. In contrast, the carboxylate group of As p83 cannot interact directly with bound substrate hut can accept a hyd rogen bond from the hydroxyl group of Thr45. To assess the role of the Thr45-Asp83 hydrogen bond in catalysis, T45G, D83A and T45G/D83A RNas e A were prepared and their abilities to catalyze the cleavage of vari ous substrates were determined. The results indicate that the side-cha in of Asp83 enhances catalysis of reactions in which uridine is bound in the B1 subsite, but that this enhancement relies on the side-chain of Thr45. In contrast, the side-chain of Asp83 does not contribute to catalysis of reactions with cytidine in the B1 subsite. Thermodynamic cycles derived from kinetic parameters for the cleavage of poly(U) ind icate that the Thr45-Asp83 interaction contributes 1.2 kcal/mol to tra nsition state stabilization, which is 0.9 kcal/mol greater than its co ntribution to ground state stabilization. Thus, like many residue-subs trate interactions, this residue to residue interaction enhances catal ysis by becoming stronger as the reaction approaches the transition st ate. (C) 1995 Academic Press Limited