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. Author manuscript; available in PMC: 2009 Jul 14.
Published in final edited form as: Biochemistry. 2009 Apr 14;48(14):3068–3077. doi: 10.1021/bi900167q

Figure 4.

Figure 4

Inhibition of E. coli LpxC by 1-68A depends upon a thiol group. (Panel A) Effect of glutathione or dithiothreitol (DTT) on 1-68A inhibition of E. coli LpxC activity. (Panel B) Reactions containing 0 μM (black squares) or 50 μM (open circles) 1-68A were initiated with 0.1 nM E. coli LpxC. Both reactions progressed for 9 min, at which point dithiothreitol was added (as indicated by the arrow) to a final concentration of 2 mM. The reduced velocity of the 0 μM reaction after 9 min is not due to dilution but rather dithiothreitol is a weak competitive inhibitor E. coli LpxC (data not shown). (Panel C) pH rate profile of wild-type E. coli LpxC specific activity (open circles) and the rate (kobs) of 1-68A inhibition (black squares). The bell-shaped profile is fit with a pK1 of 5.9 and a pK2 of 8.0. The kobs profile is fitted with a curve describing a pKa of at least 9.2. (Panel D) Reactions containing 0 μM (black squares) or 2 mM (open circles) 1-68A were initiated with 0.1 nM E. coli LpxC C207A. Both reactions progressed for 9 min, at which point dithiothreitol was added (as indicated by the arrow) to a final concentration of 2 mM. Data is representative of two separate experiments.