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- Title
Cysteine-321 of Human Brain GABA Transaminase Is Involved in Intersubunit Cross-Linking.
- Authors
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
γ-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 GABAT 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.
- Publication
Molecules & Cells (Springer Nature), 2004, Vol 18, Issue 2, p214
- ISSN
1016-8478
- Publication type
Article
- DOI
10.1016/s1016-8478(23)13104-9