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 approximately 8: for pH less-than-or-equal-to 8, the plot of log k(cat) vs pH is linear with a slope = 1.20 +/- 0.06; for pH greater-than-or-equal-to 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 greater-than-or-equal-to 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(cat)s 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 (betaVN DELTASNase) in which residues 44-49 of the OMEGA-loop (residues 43-52) have been deleted to generate a new solvent-exposed beta-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(cat)/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 OMEGA-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 betaVN DELTASNase suggest that the hydrolysis reaction is not general base catalyzed by Glu-43.