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

Published online October 31, 2004

© 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

Article

Minireview

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
Aug 31, 2022 Vol.45 No.8, pp. 513~602
COVER PICTURE
Cryo-EM structure of human porphyrin transporter ABCB6 (main figure) shows that binding of hemin (inset, magenta) in concert with two glutathione molecules (cyan) primes ABCB6 for high ATP turnover (Kim et al., pp. 575-587).

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