Bacterial and Viral Sialidases: Contribution of the Conserved Active Site Glutamate to Catalysis
| Autor: | Andrew J. Bennet, Jefferson Y. Chan, Thor J. Borgford, Jacqueline N. Watson, April Lu, Viviana C. Cerda |
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| Rok vydání: | 2011 |
| Předmět: |
Clostridium perfringens
Stereochemistry Glutamic Acid Neuraminidase Crystallography X-Ray Sialidase Micromonospora Biochemistry Catalysis Substrate Specificity Micromonospora viridifaciens Conserved sequence Residue (chemistry) Catalytic Domain Humans Enzyme kinetics Conserved Sequence biology Chemistry Deuterium Exchange Measurement Active site Catalytic cycle Influenza A virus Mutagenesis Site-Directed biology.protein Baculoviridae Cysteine |
| Zdroj: | Biochemistry. 51:433-441 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi201019n |
| Popis: | Mutagenesis of the conserved glutamic acid of influenza type A (E277) and Micromonospora viridifaciens (E260) sialidases was performed to probe the contribution of this strictly conserved residue to catalysis. Kinetic studies of the E260D and E260C M. viridifaciens mutant enzymes reveal that the overall mechanism of action has not changed. That is, the mutants are retaining sialidases in which glycosylation and deglycosylation are rate-limiting for k(cat)/K(m) and k(cat), respectively. The solvent kinetic isotope effect and proton inventory on k(cat) for the E260C mutant sialidase provide strong evidence that the newly installed cysteine residue provides little catalytic acceleration. The results are consistent with the conserved aspartic acid residue (D92) becoming the key general acid/base residue in the catalytic cycle. In addition, the E277D mutant influenza type A sialidase is catalytically active toward 4-nitrophenyl α-D-sialoside, although no measurable hydrolysis of natural substrates was observed. Thus, mutating the glutamate residue (E277) to an aspartate increases the activation free energy of hydrolysis for natural substrates by22 kJ/mol. |
| Databáze: | OpenAIRE |
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