The catalytic rate of wild type, two single (Lys 120 --> Leu, Lys 134 --> Thr), and one double (Lys 120 --> Leu-Lys 134 --> Thr) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the activity of the wild-type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK(1)) and at pH 11.3 (pK(2)) Catalytic rate measurements on single and double mutants indicate that pK, is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK(2) is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141.
Polticelli, F., Battistoni, A., O'Neill, P., Rotilio, G., Desideri, A. (1996). Identification of the residues responsible for the alkaline inhibition of Cu,Zn superoxide dismutase: A site-directed mutagenesis approach. PROTEIN SCIENCE, 5(2), 248-253.
Identification of the residues responsible for the alkaline inhibition of Cu,Zn superoxide dismutase: A site-directed mutagenesis approach
BATTISTONI, ANDREA;ROTILIO, GIUSEPPE;DESIDERI, ALESSANDRO
1996-01-01
Abstract
The catalytic rate of wild type, two single (Lys 120 --> Leu, Lys 134 --> Thr), and one double (Lys 120 --> Leu-Lys 134 --> Thr) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the activity of the wild-type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK(1)) and at pH 11.3 (pK(2)) Catalytic rate measurements on single and double mutants indicate that pK, is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK(2) is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141.File | Dimensione | Formato | |
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