期刊
BIOCHEMISTRY
卷 50, 期 14, 页码 2881-2888出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi1020259
关键词
-
资金
- Programme Interdisciplinaire du CNRS (PIE: DIALoG)
- Agence Nationale de la Recherche, ANR blanche Spinfold [ANR-09-Blan-0100, ANR-09-CP2D-06-04]
- Centre National de la Recherche Scientifique
- Agence Nationale de la Recherche (ANR) [ANR-09-BLAN-0100] Funding Source: Agence Nationale de la Recherche (ANR)
In Chlamydomonas reinhardtii, glyceraldehyde-3-phoshate dehydrogenase (GAPDH) consists of four GapA subunits. This GAPDH is not autonomously regulated, as the regulatory cysteine residues present on GapB subunits are missing in GapA subunits. The regulation of A(4) GAPDH is provided by another protein, CP12. To determine the molecular mechanisms of regulation of A4 GAPDH, we mutated three residues (R82, R190, and S195) of GApDH of C. reinhardtii. Kinetic studies of GA.PDH mutants showed the importance of residue R82 in the specificity of GAPDH for NADPH, as previously shown for the spinach enzyme. The cofactor NADPH was not stabilized through the 2'-phosphate by the serine 195 residue of the algal GAPDH, unlike the case in spinach. The mutation of R190 also led to a structural change that was not observed in the spinach enzyme. This mutation led to a loss of activity for NADPH and NADH, indicating the crucial role of this residue in maintaining the algal GAPDH structure. Finally, the interaction between GAPDH mutants and wild-type and mutated CP12 was analyzed by immunoblotting experiments, surface plasmon resonance, and kinetic studies. The results obtained with these approaches highlight the involvement of the last residue of CP12, Asp80, in modulating the activity of GA.PDH by preventing access of the cofactor NADPH to the active site. These results help us to bridge the gap between our knowledge of structure and our understanding of functional biology in GAPDH regulation.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据