MECHANISM OF THE REACTION CATALYZED BY STAPHYLOCOCCAL NUCLEASE - IDENTIFICATION OF THE RATE-DETERMINING STEP

The hydrolysis of single-stranded DNA catalyzed by wild-type staphyloc occal nuclease (SNase) and two mutants has been studied as a function of both pH and solvent viscosity. The k(cat) for wild-type SNase incre ases with pH; the slope of the plot of log k(cat) vs pH = 0.45 +/- 0.0 1. 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, t he plot of log k(cat) vs pH is linear with a slope = 1.20 +/- 0.06; fo r pH greater-than-or-equal-to 8, the slope = 0.00 +/- 0.04. The depend encies 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 s olvent viscosity; above pH 7.3, both are inversely proportional to sol vent 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 s teps (product dissociation for k(cat) and substrate binding for k(cat) /K(m)) and not an internal step (e.g., hydrolysis of the phosphodieste r bond). We have also studied the E43D mutant in which the putative ac tive-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 prop ortional to pH (slopes = 1.01 +/- 0.03 and 0.95 +/- 0.04, respectively ) and independent of solvent viscosity. At pH 9.5, the rate-determinin g 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 mutan ts of SNase with those of wild-type SNase are mechanistically uninform ative at pH values where the k(cat)s are maximal. We have begun to loc alize the structural elements responsible for the slow external step ( product dissociation) in the k(cat) for wild-type SNase by characteriz ing a mutant (betaVN DELTASNase) in which residues 44-49 of the OMEGA- loop (residues 43-52) have been deleted to generate a new solvent-expo sed 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 S Nase at the highest pH values. This behavior suggests that residues in the OMEGA-loop may be responsible for the rate-determining external s tep that characterizes the k(cat) of wild-type SNase at high pH. The d ependencies of k(cat) on pH for both wild-type SNase and betaVN DELTAS Nase suggest that the hydrolysis reaction is not general base catalyze d by Glu-43.