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Mol. Cells 2004; 18(2): 214-219

Published online January 1, 1970

© The Korean Society for Molecular and Cellular Biology

Cysteine-321 of Human Brain GABA Transaminase Is Involved in Intersubunit Cross-Linking

Chang Sik Yoon, Dae Won Kim, Sang Ho Jang, Byung Ryong Lee, Hee Soon Choi, Soo Hyun Choi, So Young Kim, Jae Jin An, Oh-Shin Kwon, Tae-Cheon Kang, Moo Ho Won, Sung-Woo Cho, Kil Soo Lee, Jinseu Park, Won Sik Eum, Soo Young Choi

Abstract

g-Aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5?dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T.

Keywords Cysteine 321 Residue; Human Brain GABA-T; Intersubunit Linkage; Neurotransmitter GABA; Site-directed Mutagenesis

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Mol. Cells 2004; 18(2): 214-219

Published online October 31, 2004

Copyright © The Korean Society for Molecular and Cellular Biology.

Cysteine-321 of Human Brain GABA Transaminase Is Involved in Intersubunit Cross-Linking

Chang Sik Yoon, Dae Won Kim, Sang Ho Jang, Byung Ryong Lee, Hee Soon Choi, Soo Hyun Choi, So Young Kim, Jae Jin An, Oh-Shin Kwon, Tae-Cheon Kang, Moo Ho Won, Sung-Woo Cho, Kil Soo Lee, Jinseu Park, Won Sik Eum, Soo Young Choi

Abstract

g-Aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5?dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T.

Keywords: Cysteine 321 Residue, Human Brain GABA-T, Intersubunit Linkage, Neurotransmitter GABA, Site-directed Mutagenesis

Mol. Cells
Nov 30, 2023 Vol.46 No.11, pp. 655~725
COVER PICTURE
Kim et al. (pp. 710-724) demonstrated that a pathogen-derived Ralstonia pseudosolanacearum type III effector RipL delays flowering time and enhances susceptibility to bacterial infection in Arabidopsis thaliana. Shown is the RipL-expressing Arabidopsis plant, which displays general dampening of the transcriptional program during pathogen infection, grown in long-day conditions.

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