Catalysis by ribonuclease A is limited by the rate of substrate association.

The value of k(cat)/K(M) for catalysis of RNA cleavage by ribonuclease (RNase) A can exceed 10(9) M(-1) s(-1) in a solution of low salt concentration. This value approaches that expected for the diffusional encounter of the enzyme and its substrate. To reveal the physicochemical constraints upon catalysis by RNase A, the effects of salt concentration, pH, solvent isotope, and solvent viscosity on catalysis were determined with synthetic substrates that bind to all of the enzymic subsites and thereby enable a meaningful analysis. The pK(a) values determined from pH-k(cat)/K(M) profiles at 0.010, 0.20, and 1.0 M NaCl are inconsistent with the known macroscopic pK(a) values of RNase A. This incongruity indicates that catalysis of RNA cleavage by RNase A is limited by the rate of substrate association, even at 1.0 M NaCl. The effect of solvent isotope and solvent viscosity on catalysis support this conclusion. The data are consistent with a mechanism in which RNase A associates with RNA in an intermediate complex, which is stabilized by Coulombic interactions, prior to the formation of a Michaelis complex. Thus, RNase A has evolved to become an enzyme limited by physics rather than chemistry, a requisite attribute of a perfect catalyst.