Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking.
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Yoon CS, Kim DW, Jang SH, Lee BR, Choi HS, Choi SH, Kim SY, An JJ, Kwon OS, Kang TC, Won MH, Cho SW, Lee KS, Park J, Eum WS, Choi SY
Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking.
Mol Cells. 2004 Oct 31;18(2):214-9.
- PubMed ID
- 15528998 [ View in PubMed]
- 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.