Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes1

FEBS Letters

Kim, Tae Ue³  Yang, Seung-Ju¹  Huh, Jae-Wan¹  Hong, Hea-Nam²  Cho, Sung-Woo¹ 

[1] Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, 388-1 Poongnap-2dong, Songpa-gu, Seoul 138-736, South Korea;Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Seoul, South Korea;Department of Biomedical Laboratory Science, College of Health Science, Yonsei University, Wonju, South Korea

关键词: Glutamate dehydrogenase;    Thermal stability;    Isozyme;    Cassette mutagenesis;    Reactive serine;    Allosteric effector;    GDH;    glutamate dehydrogenase;   

DOI  :  10.1016/S0014-5793(04)00183-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45°C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45°C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.

【 授权许可】

Unknown