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 kcat for wild-type SNase increases with pH; the slope of the plot of log kcat vs pH = 0.45 ± 0.01. The dependence of kcat/Km on pH for wild-type SNase is biphasic with a break at pH ~8: for pH ≤8, the plot of log kcat vs pH is linear with a slope = 1.20 ± 0.06; for pH ≥8, the slope = 0.00 ± 0.04. The dependencies of both kcat and kcat/Km 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 kcat and kcat/Km are external steps (product dissociation for kcat and substrate binding for kcat/Km) 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 kcat and log (kcat/Km) 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 kcats are maximal. We have begun to localize the structural elements responsible for the slow external step (product dissociation) in the kcat 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 kcat and log (kM/Km) are directly proportional to pH (slopes = 0.91 ± 0.02 and 0.78 ± 0.02, respectively) and independent of solvent viscosity, even though the kcat 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 kcat of wild-type SNase at high pH. The dependencies of kcat on pH for both wild-type SNase and βVN ΔSNase suggest that the hydrolysis reaction is not general base catalyzed by Glu-43.
ASJC Scopus subject areas