Value of General Acid–Base Catalysis to Ribonuclease A

Abstract

Bovine pancreatic ribonuclease A (RNase A) is a much studied enzyme that efficiently catalyzes the cleavage of RNA. The active site of RNase A contains two histidine residues with imidazole groups positioned to act as a general base (H12) and a general acid (H119) during catalysis of RNA cleavage. Recombinant DNA techniques were used to produce mutant enzymes in which either H12 or H119 was replaced with an alanine residue. Each mutation resulted in a 10⁴-fold decrease in the value of k_cat/K_m for cleaving either poly(C) or UpA. Thus, H12 and H119 each lower by 5–6 kcal/mol the free energy of the rate-limiting transition state during RNA cleavage. The value of k_cat/K_m for cleavage of UpOC₆H₄-p-NO₂ was decreased by 10⁴-fold by replacing H12 but was unaffected by replacing H119. This result provides the first direct evidence that H119 acts as a general acid during catalysis by RNase 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, k_cat/K_m, 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.

Table 1.

Steady-State Kinetic Parameters for Cleavage of Ribonucleotides by Wild-Type and Mutant Ribonucleases^a

RNase A	substrateb	kcat (s–1)	Km (mM)	kcat/Km (M–1 s–1)	$\frac{{(k_{\mathrm{cat}}∕K_{\mathrm{m}})}^{\mathrm{mutant}}}{{(k_{\mathrm{cat}}∕K_{\mathrm{m}})}^{\text{wild-type}}}$
wild-type	poly(C)	(4.1 ± 0.1) × 102 c	0.034 ± 0.002c	(1.5 ± 0.1) × 107 c	1.0
H12A	poly(C)	0.073 ± 0.006	0.105 ± 0.025	(7.3 ± 0.2) × 102	(4.9 ± 0.9) × 10–5
H119A	poly(C)	0.24 ± 0.03	0.21 ± 0.03	(1.1 ± 0.1) × 103	(7.3 ± 0.8) × 10–5
wild-type	UpA	(1.40 ± 0.15) × 103 c	0.62 ± 0.09c	(2.3 ± 0.4) × 106 c	1.0
H12A	UpA	0.15 ± 0.02	0.86 ± 0.17	(1.7 ± 0.1) × 102	(7.4 ± 1.4) × 10–5
H119A	UpA	0.76 ± 0.10	0.80 ± 0.15	(9.5 ± 0.5) × 102	(4.1 ± 0.7) × 10–4
wild-type	UpOC6H4-p-NO2	18.8 ± 0.6	0.33 ± 0.05	(5.7 ± 0.6) × 104	1.0
H12A	UpOC6H4-p-NO2	0.0029 ± 0.0001	0.275 ± 0.041	(1.1 ± 0.1) × 101	(1.9 ± 0.3) × 10–4
H119A	UpOC6H4-p-NO2	27 ± 1	0.76 ± 0.08	(3.6 ± 0.1) × 104	0.63 ± 0.11

^aAll reactions were performed at 25 °C in 50 mM MES buffer, pH 6.0, containing 0.1 M NaCl. Steady-state kinetic parameters were determined by fitting the initial velocity data to a hyperbolic curve using the program HYPERO.²²

^bCleavage of poly(C) and UpOC₆H₄-p-NO₂ were monitored at 250 nm (Δε₂₅₀ = 2380 M^–1 cm^–1) and 330 nm (Δε₃₃₀ = 6200 M^–1 cm^–1), respectively; cleavage of UpA was monitored at 265 nm in the presence of excess adenosine deaminase²³ (Δε₂₆₅ = –6000 M^–1 cm^–1).

^cData from ref 8.

Figure 2.

Values of k_cat/K_m for the cleavage reaction catalyzed by wild-type and mutant ribonucleases.

The value of the imidazole group of H119 to catalysis depends on the pK_a 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 pK_a ≈ 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 pK_a = 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 $k_{\mathrm{wt}}∕k_{\mathrm{H}12\mathrm{A}}={(K_{\mathrm{a}}^{{\mathrm{H}}_3{\mathrm{O}}^{+}}∕K_{\mathrm{a}}^{\mathrm{H}12})}^{\beta }$ and $k_{\mathrm{wt}}∕k_{\mathrm{H}119\mathrm{A}}={(K_{\mathrm{a}}^{\mathrm{H}119}∕K_{\mathrm{a}}^{{\mathrm{H}}_2\mathrm{O}})}^{\alpha }$, where $\mathrm{p}{K_{\mathrm{a}}}^{\mathrm{H}12}=5.8$ and $\mathrm{p}{K_{\mathrm{a}}}^{\mathrm{H}119}=6.2$¹⁷ and $\mathrm{p}{K}_{\mathrm{a}}^{{\mathrm{H}}_3{\mathrm{O}}^{+}}=-1.7$ and $\mathrm{p}{K}_{\mathrm{a}}^{{\mathrm{H}}_2\mathrm{O}}=15.7$. The Brønsted equation therefore predicts that general base catalysis provides a 10^7.5β-fold rate enhancement, and general acid catalysis provides a 10^9.5α-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 ΔG_app = 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.