TAILIEUCHUNG - Báo cáo khoa học: 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-dimethylamino-azobenzene-4-iodoacetamide (DABIA) resulted in a time-dependent loss of enzymatic activity. This inactivation followed pseudofirst-order kinetics with a second-order rate constant of 168M )1 Æmin )1 .The spectrumofDABIA-labeled enzyme showed a characteristic peak of the DABIA alkyl-ated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. | Eur. J. Biochem. 271 4903-4908 2004 FEBS 2004 doi Brain succinic semialdehyde dehydrogenase Reactions of sulfhydryl residues connected with catalytic activity Bvuna Rvona Lee1. Dae Won Kim1 Jouna-Woo Nona2. Won Sik Eum1. Hee Soon Choi1 Soo Hvun Choi1 So Young Kim1 Jae Jin An1 Jee-Yin Ahn1 Oh-Shin Kwon3 Tae-Cheon Kang4 Moo Ho Won4 Sung-Woo Cho5 Kil Soo Lee1 Jinseu Park1 and Soo Young Choi1 1 Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology Hallym University Chunchon Korea department of Molecular and Cellular Biochemistry The Ohio State University Columbus OH USA 3 Department of Biochemistry Kyungpook National University Taegu Korea department of Anatomy College of Medicine Hallym University Chunchon Korea 5Department of Biochemistry and Molecular Biology University of Ulsan College of Medicine Seoul Korea Incubation of an NAD -dependent succinic semialdehyde dehydrogenase from bovine brain with 4-dimethylamino-azobenzene-4-iodoacetamide DABIA resulted in a timedependent loss of enzymatic activity. This inactivation followed pseudo first-order kinetics with a second-order rate constantof 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 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

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