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Value of general Acid-base catalysis to ribonuclease a.

Bovine pancreatic ribonuclease A (RNase A; E.C. 3.1.27.5) has been one of the most studied of all enzymes.1 RNase A efficiently catalyzes the cleavage of RNA.2 Early X-ray diffraction analyses revealed that the active site of RNase A contains two histidine residues, histidine 12 (H12) and histidine 119 (H119).3 The results of chemical modification4 and pH–rate5 studies are consistent with an enzymatic reaction mechanism in which the rate-limiting transition state for RNA cleavage is similar to that shown in Figure 1. In this mechanism, the imidazole side chain of H12 acts as a general base by deprotonating the 2′ oxygen, and that of H119 acts as a general acid by protonating the 5″ oxygen. These two residues have evoked much interest in bioorganic chemistry,6 as well as in protein chemistry and enzymology.1 Indeed, no residue other than H12 and H119 need be invoked to explain the classic bell shape of the pH–rate profile5 for catalysis by this enzyme. Here, we report the explicit value of this general acid and this general base to catalysis by RNase A. 
 
 
 
Figure 1 
 
Putative structure of the transition state for the RNase A-catalyzed cleavage of RNA. Proposed interactions with H12 (★) and H119 (☆) are indicated. 
 
 
 
We used recombinant DNA techniques to produce mutant ribonucleases in which either H12 or H119 was changed to an alanine residue.7 This change effectively substitutes a proton for the imidazole group of each residue. We then determined the ability of the resulting mutant enzymes, H12A RNase A and H119A RNase A, to catalyze the cleavage of three phosphodiester substrates: polycytidylic acid [poly(C)], uridylyl(3′→5′)adenosine (UpA), and uridine 3′-(p-nitrophenylphosphate) (UpOC6H4-p-NO2).9 
 
The values of the steady-state kinetic parameters for cleavage of poly(C), UpA, and UpOC6H4-p-NO2 by the wild-type and mutant ribonucleases are given in Table 1 and Figure 2. The second-order rate constant, kcat/Km, is proportional to the association constant of an enzyme and the rate-limiting transition state during catalysis.10 As shown in Figure 2, eliminating the imidazole group of H12 decreased the affinity of the enzyme for this transition state by 104-fold during cleavage of poly(C), UpA, and UpOC6H4-p-NO2.11 Eliminating the imidazole group of H119 decreased this affinity by 104-fold during cleavage of poly(C) and by almost 104-fold during cleavage of UpA.12 In contrast, this change had no significant effect on the rate of cleavage of UpOC6H4-p-NO2. 
 
 
 
Figure 2 
 
Values of kcat/Km for the cleavage reaction catalyzed by wild-type and mutant ribonucleases. 
 
 
 
 
 
Table 1 
 
Steady-State Kinetic Parameters for Cleavage of Ribonucleotides by Wild-Type and Mutant Ribonucleasesa 
 
 
 
The value of the imidazole group of H119 to catalysis depends on the pKa of the conjugate acid of the leaving groups. Cleavage of poly(C) and UpA is accelerated dramatically by the side chain of H119. The nucleotide and nucleoside leaving groups in these substrates have conjugate acids with pKa ≈ 14.8.13 In contrast, the cleavage of UpOC6H4-p-NO2 is unaffected by the side chain of H119. The p-nitrophenolate leaving group in substrate has a conjugate acid with pKa = 7.14.14 Together, these data provide the first direct evidence that the role of H119 is to protonate the leaving group during RNA cleavage. This result also illustrates how a capable catalyst for cleavage of an activated model substrate (e.g., UpOC6H4-p-NO2) can lack a component important for cleavage of an unactivated substrate (e.g., RNA). 
 
The results for UpOC6H4-p-NO2 illuminate the mechanism of catalysis by RNase A. Breslow has proposed that RNase A catalyzes RNA cleavage via a phosphorane intermediate.6b In the Breslow mechanism, H119 is proposed to both protonate a non-bridging oxygen of the phosphate anion and deprotonate this same oxygen in the phosphorane intermediate.15 Yet, wild-type and H119A RNase A cleaved UpOC6H4-p-NO2 at the same rate (Figure 2). Thus our data argue against the participation of H119 in the formation of a phosphorane, at least during the cleavage of UpOC6H4-p-NO2.16 
 
The steady-state kinetic parameters of wild-type, H12A, and H119A RNase A are consistent with H12 acting as a general base and show that H119 acts as a general acid during the cleavage of RNA. Further, the observed rate enhancements agree with those expected for acid–base catalysis by H12 and H119. For example, suppose a water molecule replaces the imidazole group in the mutant enzymes such that the interactions marked by ★ or ☆ in Figure 1 are now to the oxygen or a hydrogen, respectively, of H2O. The rate enhancements then derived from the Brfnsted equation are kwt∕kH12A=(KaH3O+∕KaH12)β and kwt∕kH119A=(KaH119∕KaH2O)α, where pKaH12=5.8 and pKaH119=6.217 and pKaH3O+=−1.7 and pKaH2O=15.7. The Bronsted equation therefore predicts that general base catalysis provides a 107.5β-fold rate enhancement, and general acid catalysis provides a 109.5α-fold rate enhancement. Values of α and β tend to be approximately 0.5 for proton transfers between oxygen and nitrogen.18 Thus, the rate enhancements predicted with this simple model are similar to those observed (Figure 2). 
 
The side chains of H12 and H119 each bind to the rate-limiting transition state during RNA cleavage with an apparent free energy of ΔGapp = 5–6 kcal/mol.19 This free energy is related20 to the strength of the interaction marked by ★ or ☆ in Figure 1. For many reasons, however, neither of these interactions can be assigned an explicit free energy. For example, mutagenesis may have altered the structure of the transition state or its position on the reaction coordinate.10,18,20 Also, catalysis by wild-type RNase A may be limited in part by a diffusive step rather than a chemical interconversion.21 Studies to illuminate these and other aspects of catalysis by this venerable enzyme are underway.