Bovine pancreatic ribonuclease (\mathrm{A}) (RNase A) catalyzes the cleavage of the (\mathrm{P}-\mathrm{O}^{5}) bond in RNA. Although this enzyme has been the object of much seminal work in biological chemistry, the nature of its rate-limiting transition state and its catalytic rate enhancement had been unknown. Here, the value of (k_{\text {cal }} / K_{m}) for the cleavage of UpA by wild-type RNase A was found to be inversely related to the concentration of added glycerol. In contrast, the values of (k_{\mathrm{ca}} / K_{m}) for the cleavage of UPA by a sluggish mutant of RNase A and the cleavage of the poor substrate (\mathrm{UpOC}{6} \mathrm{H}{4}-p-\mathrm{NO}{2}) by wild-type (\mathrm{RNase} A) were found to be independent of glycerol concentration. Yet, the values of (k{\mathrm{ca}} / K_{m}) for UPA cleavage by the wild-type and mutant enzymes were found to have an identical dependence on the concentration of added sucrose. Although both glycerol and sucrose are viscogenic, only glycerol interacts strongly with single-stranded nucleic acids. Catalysis of UpA cleavage by RNase A is therefore limited by substrate desolvation. The rate of UpA cleavage by (\mathrm{RNase} \mathrm{A}) is maximal at (\mathrm{pH} 6.0), where (k_{\text {cat }}=1.4 \times 10^{3} \mathrm{~s}^{-1}) and (k_{\text {cat }} / K_{m}=2.3 \times 10^{6} \mathrm{M}^{1} \mathrm{~s}^{-1}) at (25^{\circ} \mathrm{C}). At pH 6.0 and (25^{\circ} \mathrm{C}), the uncatalyzed rate of (\left[5,6^{-3} \mathrm{H}\right] \mathrm{Up}\left[3,5,8^{-3} \mathrm{H}\right] \mathrm{A}) cleavage was found to be (k_{\text {uncal }}=5 \times 10^{-9} \mathrm{~s}^{-1}) (\left(t_{1 / 2}=4\right.) years). Thus, RNase A enhances the rate of UPA cleavage by (3 \times 10^{11})-fold by binding to the transition state for (\mathrm{P}-\mathrm{O}^{\mathrm{s}}) bond cleavage with a dissociation constant of (<2 \times 10^{-15}) M. 1995 Academic Press, Inc.