Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A |
| |
Authors: | Chong Yuan Hongyu Hu Genjun Xu |
| |
Institution: | (1) Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200031 Shanghai, China |
| |
Abstract: | 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. |
| |
Keywords: | lactate dehydrogenase A N-terminal residues size-exclusion chromatograph tetramer stability site-directed mutagenesis unfolding experiments subunit interaction |
本文献已被 CNKI SpringerLink 等数据库收录! |
| 点击此处可从《中国科学:生命科学英文版》浏览原始摘要信息 |
| 点击此处可从《中国科学:生命科学英文版》下载免费的PDF全文 |
|