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pH-dependence of active site point mutants. (A) The D99A LpxK mutant was assayed in a triple-buffer system (100 mM sodium acetate, 50 mM Tris, 50 mM bis-Tris) at the indicated pH values and specific activity assessed. A pKa of 5.8 ± 0.1 and pKb of 9.6 ± 0.1 were calculated based on the data. (B) In the same manner, the pH dependence of the H261A mutant was analyzed resulting in pKa and pKb values of 6.0 ± 0.1 and 9.9 ± 0.2, respectively.
