title: Site-directed mutagenesis of human brain GABA transaminase: lysine-357 is involved in cofactor binding at the active site.
abstract: gamma-Aminobutyrate transaminase (GABA-T), a key enzyme of the GABA shunt, converts the major inhibitory neurotransmitter, GABA, to succinic semialdehyde. Although GABA-T is a pivotal factor implicated in the pathogenesis of various neurological disorders, its function remains to be elucidated. In an effort to clarify the structural and functional roles of specific lysyl residue in human brain GABA-T, we constructed human brain GABA-T mutants, in which the lysyl residue at position 357 was mutated to various amino acids including asparagine (K357N). The purified mutant GABA-T enzymes displayed neither catalytic activity nor absorption bands at 330 and 415 nm that are characteristic of pyridoxal-5'-phosphate (PLP) covalently linked to the protein. The wild type apoenzyme reconstituted with exogenous PLP had catalytic activity, while the mutant apoenzymes did not. These results indicate that lysine 357 is essential for catalytic function, and is involved in binding PLP at the active site.
authors.affiliation: Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
authors.forename: Dae Won
authors.initials: DW
authors.lastname: Kim
authors.forename: Chang Sik
authors.initials: CS
authors.lastname: Yoon
authors.forename: Won Sik
authors.initials: WS
authors.lastname: Eum
authors.forename: Byung Ryong
authors.initials: BR
authors.lastname: Lee
authors.forename: Jae Jin
authors.initials: JJ
authors.lastname: An
authors.forename: Sun Hwa
authors.initials: SH
authors.lastname: Lee
authors.forename: Seung Ree
authors.initials: SR
authors.lastname: Lee
authors.forename: Jee-Yin
authors.initials: JY
authors.lastname: Ahn
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: Soo Young
authors.initials: SY
authors.lastname: Choi
mesh_terms: D000612:4-Aminobutyrate Transaminase / Q000235:genetics* / Q000502:physiology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry
chemical_list: D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000612:4-Aminobutyrate Transaminase; D008239:Lysine