Site-directed mutagenesis of human dihydrolipoamide dehydrogenase: role of lysine-54 and glutamate-192 in stabilizing the thiolate-FAD intermediate
| Autor: | Young Soo Hong, Nataraj N. Vettakkorumakankav, Mulchand S. Patel, Te-Chung Liu, Lioubov G. Korotchkina |
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| Rok vydání: | 1999 |
| Předmět: |
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Molecular Dihydrolipoamide Protein Conformation Mutant Genetic Vectors Glutamic Acid Biology In Vitro Techniques Electron Transport chemistry.chemical_compound Enzyme Stability medicine Escherichia coli Humans Enzyme kinetics Site-directed mutagenesis DNA Primers Dihydrolipoamide Dehydrogenase Dihydrolipoamide dehydrogenase Base Sequence Circular Dichroism Lysine Mutagenesis NAD Recombinant Proteins Kinetics Spectrometry Fluorescence chemistry Biochemistry Lipoamide Flavin-Adenine Dinucleotide Mutagenesis Site-Directed NAD+ kinase Biotechnology medicine.drug |
| Zdroj: | Protein expression and purification. 16(1) |
| ISSN: | 1046-5928 |
| Popis: | The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate-FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54-K53S54 (SK-KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK-KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein-FAD interaction. The specific activities of all mutant E3s were markedly decreased ( |
| Databáze: | OpenAIRE |
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