Limits to catalysis by ribonuclease A

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-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(cat)/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(cat)/K-m for the cleavage of UpA by a sluggish mutant of RNase A and the cleavage of the poor substrate UpOC(6)H(4)-p-NO2 by wild-type RNase A were found to be independent of glycerol concentration. Yet, the values of k(cat)/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 RNase A is maximal at pH 6.0, where k(cat) = 1.4 X 10(3) s(-1) and k(cat)/K-m = 2.3 X 10(6) M(-1) s(-1) at 25 degrees C. At pH 6.0 and 25 degrees C, the uncatalyzed rate of [5,6-H-3]Up[3,5,8-H-3]A cleavage was found to be k(uncat) = 5 X 10(-9) s(-1) (t(1/2) = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 X 10(11)-fold by binding to the transition state for P-O-5' bond cleavage with a dissociation constant of <2 X 10(-15) M. (C) 1995 Academic Press, Inc.