2,4-dinitrophenyl 2-deoxy-2-fluoro

The canonical mechanism shown in Figure 5.32 requires the same carboxylate group which acts as a general acid in the first step to act as a general base in the second. Its pAa must, therefore, decrease between the free enzyme and the glycosyl-enzyme intermediate. Direct NMR observation of active site carboxy-lates in the GH 11 p-xylanase of B. circulans already discussed in Section 5.4.2 indicated that the acid-base catalytic Glu 172 had a (microscopic) pAa of 6.7 in the free enzyme and 4.2 in the 2-deoxy-2-fluoro-a-xylobiosyl enzyme generated from the Withers inactivator 2,4-dinitrophenyl 2 -deoxy-2 -fluoro-p-xylobioside. ... [Pg.387]

Given the similarity of the Km values measured for the mutants to those of the wild-type enzyme, it was necessary to further investigate the possibility that residual activity of the active-site mutants arises from traces of wild-type enzyme, either by contamination during the purification process, by spontaneous deamidation or translational misreading (31, 32). Active site titration with the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro P-cellobioside... [Pg.213]

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