Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A
| Autor: | Chong Yuan, Hongyu Hu, Genjun Xu |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
chemistry.chemical_classification
education.field_of_study Aqueous solution Protein subunit Lactate dehydrogenase A Mutant Oligomer General Biochemistry Genetics and Molecular Biology chemistry.chemical_compound Enzyme chemistry Tetramer Biochemistry General Agricultural and Biological Sciences Site-directed mutagenesis education General Environmental Science |
| Zdroj: | Science in China. Series C, Life sciences. 44(6) |
| ISSN: | 1006-9305 |
| Popis: | Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an α-helix and a β-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and Ile8Glu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6–0.8 mol/L GdmCl. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCl (0.2 mol/L). In aqueous solution, the Ile8Glu variant exists predominantly in the dimeric form with increased KM and decreasedk cat as compared with those of WT and L3P. However, the activity of Ile8Glu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less stable than WT in oligomer structure. Results also support the fact that some residues in the N-terminal arm, especially the Leu8 in the β-structure, contribute the important binding energies to the dimerization of dimers, which might affect the assembly of the enzyme as well as the catalytic function. |
| Databáze: | OpenAIRE |
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