LIMITS TO CATALYSIS BY RIBONUCLEASE-A

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of t he P-O-5' bond in RNA. Although this enzyme has been the object of muc h seminal work in biological chemistry, the nature of its rate-limitin g 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 R Nase 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 U pA by a sluggish mutant of RNase A and the cleavage of the poor substr ate UpOC(6)H(4)-p-NO2 by wild-type RNase A were found to be independen t of glycerol concentration. Yet, the values of k(cat)/K-m for UpA cle avage by the wild-type and mutant enzymes were found to have an identi cal dependence on the concentration of added sucrose. Although both gl ycerol and sucrose are viscogenic, only glycerol interacts strongly wi th single-stranded nucleic acids. Catalysis of UpA cleavage by RNase A is therefore limited by substrate desolvation. The rate of UpA cleava ge by RNase A is maximal at pH 6.0, where k(cat) = 1.4 X 10(3) s(-1) a nd 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 cleavag e 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 bind ing to the transition state for P-O-5' bond cleavage with a dissociati on constant of <2 X 10(-15) M. (C) 1995 Academic Press, Inc.