Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking.
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.
Yoon
Chang Sik
CS
Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
Kim
Dae Won
DW
Jang
Sang Ho
SH
Lee
Byung Ryong
BR
Choi
Hee Soon
HS
Choi
Soo Hyun
SH
Kim
So Young
SY
An
Jae Jin
JJ
Kwon
Oh-Shin
OS
Kang
Tae-Cheon
TC
Won
Moo Ho
MH
Cho
Sung-Woo
SW
Lee
Kil Soo
KS
Park
Jinseu
J
Eum
Won Sik
WS
Choi
Soo Young
SY
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
D003432:Cross-Linking Reagents; D004220:Disulfides; D021122:Protein Subunits; D004228:Dithionitrobenzoic Acid; D000612:4-Aminobutyrate Transaminase; D003545:Cysteine