Mechanism of the Reaction Catalyzed by Staphylococcal Nuclease: Identification of the Rate-Determining Step

The hydrolysis of single-stranded DNA catalyzed by wild-type staphylococcal nuclease (SNase) and two mutants has been studied as a function of both pH and solvent viscosity. The k_cat for wild-type SNase increases with pH; the slope of the plot of log k_cat vs pH = 0.45 ± 0.01. The dependence of k_cat/K_m on pH for wild-type SNase is biphasic with a break at pH ~8: for pH ≤8, the plot of log k_cat vs pH is linear with a slope = 1.20 ± 0.06; for pH ≥8, the slope = 0.00 ± 0.04. The dependencies of both k_cat and k_cat/K_m on solvent viscosity are also pH-dependent: below pH 7.3, both kinetic parameters are independent of solvent viscosity; above pH 7.3, both are inversely proportional to solvent viscosity. Thus, at pH 9.5, where SNase is routinely assayed, the rate-determining steps for both k_cat and k_cat/K_m are external steps (product dissociation for k_cat and substrate binding for k_cat/K_m) and not an internal step (e.g., hydrolysis of the phosphodiester bond). We have also studied the E43D mutant in which the putative active-site general basic catalyst Glu-43 is replaced with Asp. From pH 7.5 to pH 9.5, both log k_cat and log (k_cat/K_m) are directly proportional to pH (slopes = 1.01 ± 0.03 and 0.95 ± 0.04, respectively) and independent of solvent viscosity. At pH 9.5, the rate-determining step is an internal step. Since the rate-determining steps differ at pH values ≥7.5, we conclude that comparisons of the numerical values of the kinetic parameters of active-site mutants of SNase with those of wild-type SNase are mechanistically uninformative at pH values where the k_cats are maximal. We have begun to localize the structural elements responsible for the slow external step (product dissociation) in the k_cat for wild-type SNase by characterizing a mutant (βVN ΔSNase) in which residues 44–49 of the Ω-loop (residues 43–52) have been deleted to generate a new solvent-exposed β-turn; in addition, Gly-50 and Val-51 have been replaced with Val and Asn, respectively. From pH 7 to pH 10, both log k_cat and log (k_M/K_m) are directly proportional to pH (slopes = 0.91 ± 0.02 and 0.78 ± 0.02, respectively) and independent of solvent viscosity, even though the k_cat of this mutant approaches that of wild-type SNase at the highest pH values. This behavior suggests that residues in the Ω-loop may be responsible for the rate-determining external step that characterizes the k_cat of wild-type SNase at high pH. The dependencies of k_cat on pH for both wild-type SNase and βVN ΔSNase suggest that the hydrolysis reaction is not general base catalyzed by Glu-43.