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 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 approximately 8: for pH < or = 8, the plot of log kcat vs pH is linear with a slope = 1.20 +/- 0.06; for pH > or = 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hale
S P
SP
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Poole
L B
LB
Gerlt
J A
JA
D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009691:Nucleic Acid Denaturation; D014783:Viscosity; D014961:X-Ray Diffraction
D004277:DNA, Single-Stranded; D008836:Micrococcal Nuclease