Abstract
The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn 2+, Mg 2+, Co 2+, and Zn 2+ shift the optimal pH of phosphatase activity to 6.5 while Be 2+ and Ca 2+ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me 2+ ions, Mn 2+, Co 2+, Zn 2+, and Fe 2+ decreased the K m of histidinol phosphate at pH 6.5 from 5.5 m m (without Me 2+) to 0.14 m m. Ni 2+ and Be 2+ increased the K m to 22.2 and 25.0 m m, respectively, and Ca 2+ and Mg 2+ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn 2+. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn 2+ to the enzyme before the addition of the substrate, histidinol phosphate. A k imn 2+ of 4.3 μ m was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me 2+ ions, Mg 2+ being the most efficient followed by Mn 2+. Prolonged incubation with EDTA led to irreversible inactivation.
Published Version
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