title: Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking.
abstract: Gamma-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 cent-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.
authors.affiliation: Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
authors.forename: Chang Sik
authors.initials: CS
authors.lastname: Yoon
authors.forename: Dae Won
authors.initials: DW
authors.lastname: Kim
authors.forename: Sang Ho
authors.initials: SH
authors.lastname: Jang
authors.forename: Byung Ryong
authors.initials: BR
authors.lastname: Lee
authors.forename: Hee Soon
authors.initials: HS
authors.lastname: Choi
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Choi
authors.forename: So Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Jae Jin
authors.initials: JJ
authors.lastname: An
authors.forename: Oh-Shin
authors.initials: OS
authors.lastname: Kwon
authors.forename: Tae-Cheon
authors.initials: TC
authors.lastname: Kang
authors.forename: Moo Ho
authors.initials: MH
authors.lastname: Won
authors.forename: Sung-Woo
authors.initials: SW
authors.lastname: Cho
authors.forename: Kil Soo
authors.initials: KS
authors.lastname: Lee
authors.forename: Jinseu
authors.initials: J
authors.lastname: Park
authors.forename: Won Sik
authors.initials: WS
authors.lastname: Eum
authors.forename: Soo Young
authors.initials: SY
authors.lastname: Choi
mesh_terms: D000612:4-Aminobutyrate Transaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001921:Brain; D002384:Catalysis; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D021122:Protein Subunits
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D021122:Protein Subunits; D004228:Dithionitrobenzoic Acid; D000612:4-Aminobutyrate Transaminase; D003545:Cysteine