| Sialidases (neuraminidases, EC 3.2.1.18) catalyze the hydrolysis of sialic acid from glycoconjugates, and are involved in the pathogenesis of many human diseases. The long-term objectives of the studies described herein are to (1) dissect sialidase catalytic mechanism, (2) employ knowledge of mechanism in the design improved inhibitors, and (3) explore the use of mutant sialidases for transglycosylation. We have expressed and purified a viral sialidase and a bacterial sialidase. Using site-directed mutagenesis, numerous substitutions were made in the enzyme active site in order to probe the function of three catalytic residues.; The E277D mutation in the influenza A/Tokyo/3/67 sialidase ( i.e., one of three putative catalytic residues) resulted in a 10 2-fold decrease in catalytic activity, suggesting that while important to catalysis, this residue likely does not act as a nucleophile. For the M. viridifaciens sialidase eight mutations at the remaining two positions yielded enzymes with reduced activity.; Extensive kinetic studies with the wild-type, D92G and Y370D M. viridifaciens enzymes were performed to investigate the effects of pH and leaving group ability on kinetic parameters k cat and kcat/Km. The pH profiles for D92G and Y370D were qualitatively similar to the wild type, and indicated that mutant Y370D is more active when the introduced aspartic acid is protonated. Bronsted plots were flat for the wild type enzyme, indicating that cleavage of the glycosidic bond is not rate-limiting. In contrast, Bronsted plots for the mutants D92G and Y370D revealed a change in rate-limiting step, where chemistry becomes rate-determining. Consequently, hydrolysis of natural substrates (i.e., sialyl-lactose) was reduced by 104-fold and 106-fold for D92G and Y370D, respectively. 1H-NMR spectroscopy demonstrated that the wild-type and D92G are both retaining glycosidases. In contrast, the Y370A, Y370D and Y370G mutants catalyze the hydrolysis of sialosides with an inversion of configuration. To the best of our knowledge, these are the first known inverting sialidases. These results are consistent with the replacement of the catalytic nucleophile (tyrosine) and the general-acid residue (aspartic acid). Moreover, since D92G has a high catalytic activity with activated substrates, it has potential to be used as a transglycosylation catalyst because the coupled product cannot be degraded. |
