Brain succinic semialdehyde dehydrogenase. Reactions of sulfhydryl residues connected with catalytic activity.
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.
Lee
Byung Ryong
BR
Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon, Korea.
Kim
Dae Won
DW
Hong
Joung-Woo
JW
Eum
Won Sik
WS
Choi
Hee Soon
HS
Choi
Soo Hyun
SH
Kim
So Young
SY
An
Jae Jin
JJ
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
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
D013438:Sulfhydryl Compounds; C038560:4-dimethylaminoazobenzene-4'-iodoacetamide; D004124:p-Dimethylaminoazobenzene; D000445:Aldehyde Oxidoreductases; D050644:Succinate-Semialdehyde Dehydrogenase