title: Brain succinic semialdehyde dehydrogenase. Reactions of sulfhydryl residues connected with catalytic activity.
abstract: Incubation of an NAD+-dependent succinic semialdehyde dehydrogenase from bovine brain with 4-dimethylaminoazobenzene-4-iodoacetamide (DABIA) resulted in a time-dependent loss of enzymatic activity. This inactivation followed pseudo first-order kinetics with a second-order rate constant of 168 m(-1).min(-1). The spectrum of DABIA-labeled enzyme showed a characteristic peak of the DABIA alkylated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. A linear relationship was observed between DABIA binding and the loss of enzyme activity, which extrapolates to a stoichiometry of 8.0 mol DABIA derivatives per mol enzyme tetramer. This inactivation was prevented by preincubating the enzyme with substrate, succinic semialdehyde, but not by preincubating with coenzyme NAD+. After tryptic digestion of the enzyme modified with DABIA, two peptides absorbing at 436 nm were isolated by reverse-phase HPLC. The amino acid sequences of the DABIA-labeled peptides were VCSNQFLVQR and EVGEAICTDPLVSK, respectively. These sites are identical to the putative active site sequences of other brain succinic semialdehyde dehydrogenases. These results suggest that the catalytic function of succinic semialdehyde dehydrogenase is inhibited by the specific binding of DABIA to a cysteine residue at or near its active site.
authors.affiliation: Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon, Korea.
authors.forename: Byung Ryong
authors.initials: BR
authors.lastname: Lee
authors.forename: Dae Won
authors.initials: DW
authors.lastname: Kim
authors.forename: Joung-Woo
authors.initials: JW
authors.lastname: Hong
authors.forename: Won Sik
authors.initials: WS
authors.lastname: Eum
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: 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: D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002417:Cattle; D007700:Kinetics; D050644:Succinate-Semialdehyde Dehydrogenase; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D013438:Sulfhydryl Compounds; C038560:4-dimethylaminoazobenzene-4'-iodoacetamide; D004124:p-Dimethylaminoazobenzene; D000445:Aldehyde Oxidoreductases; D050644:Succinate-Semialdehyde Dehydrogenase