FIGURE 3.
pH-rate profiles indicate that Glu-51 and Lys-53 act as the general base and acid, respectively, in the dehydration step of IGP production. Shown are the pH-rate profiles for kcat at 25 °C (left) and 75 °C (right) for WT ssIGPS (A) and variants K53R (B) and E51Q (C). Km values were pH-independent. Note the logarithmic scale on the y axes of these plots. In the K53R variant, the pKa2 is substantially larger, as expected if the residue at position 53 acts as a general acid. The lack of the ascending limb in the pH-rate profile of the E51Q variant is consistent with Glu-51 acting as a general base in ssIGPS catalysis. Both the K53R and E51Q variants show shifts in their other pKa values. These effects are likely caused by changes to the local microenvironment. For example, the loss of the negatively charged carboxylate group of Glu-51 would tend to lower the pKa of the nearby side chain of Lys-53.