Site-directed mutagenesis of human brain GABA transaminase: lysine-357 is involved in cofactor binding at the active site.
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.
Kim
Dae Won
DW
Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
Yoon
Chang Sik
CS
Eum
Won Sik
WS
Lee
Byung Ryong
BR
An
Jae Jin
JJ
Lee
Sun Hwa
SH
Lee
Seung Ree
SR
Ahn
Jee-Yin
JY
Kwon
Oh-Shin
OS
Kang
Tae-Cheon
TC
Won
Moo Ho
MH
Cho
Sung-Woo
SW
Lee
Kil Soo
KS
Park
Jinseu
J
Choi
Soo Young
SY
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
D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000612:4-Aminobutyrate Transaminase; D008239:Lysine