| Abstract: | The mechanism of the Cu-Zn-containing superoxide dismutase (SD) was studied using a stopped-flow spectrophotometric system capable of forming aqueous solutions of O2- having initial concentrations up to approximately 5 mM. By lowering the temperature to 5.5 degrees C, we were able to observe saturation of the enzyme. At 5.5 degrees C and pH 9.3, the Michaelis-Menten parameters extracted from the kinetic traces were turnover number (TN) approximately 1 X 10(6) s-1, Km approximately 3.5 X 10(-3) M. Under our conditions, the average rate at which O-2 binds to the active site, TN/Km is 0.26 X 10(9) M-1 s-1. TN was decreased in the presence of D2O, and a solvent isotope effect of TNH/TND approximately 3.6 was measured while TN/Km was essentially unaffected by D2O. TN was increased by the presence of the general acid, ND4+. These observations, by analogy to earlier work with Fe X SD from Escherichia coli (Bull, C., and Fee, J. A. (1985) J. Am. Chem. Soc. 107, 3295-3304), suggest that H2O serves to donate the protons required to form product H2O2. Values of Km and TN for the zinc-deficient enzyme were found to be approximately a factor of 2 less than those obtained for the holoenzyme under identical experimental conditions, whereas TN/Km was largely unchanged. The imidazolate bridge is thus not essential for catalytically competent extraction of a proton from the solvent. |
