Limits to Catalysis by Ribonuclease A

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O5′ 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 kcat/Km 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 kcat/Km for the cleavage of UpA by a sluggish mutant of RNase A and the cleavage of the poor substrate UpOC6H4-p-NO2 by wild-type RNase A were found to be independent of glycerol concentration. Yet, the values of kcat/Km 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 RNase A is maximal at pH 6.0, where kcat = 1.4 × 103 s−1 and kcat/Km = 2.3 × 106 M−1 s−1 at 25°C. At pH 6.0 and 25°C, the uncatalyzed rate of [5,6-3H]Up[3,5,8-3H]A cleavage was found to be kuncat = 5 × 10−9 s−1 (t12 = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 × 1011-fold by binding to the transition state for P-O5′ bond cleavage with a dissociation constant of <2 × 10−15 M.