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KMID : 0578319950050060611
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Molecules and Cells
1995 Volume.5 No. 6 p.611 ~ p.617
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Bovine Brain succinic Semialdehyde Dehydrogenase
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Lee, Byung Ryong
Hong, Joung Woo/Yoo, Byung Kwon/lee, Su Jin/Cho, Sung-Woo/Choi, Soo Young
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Abstract
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The enzyme succinic semialdehyde dehydrogenase from bovine brain has been 1,300-fold purified by a combination of CM-Sepharose, Blue-Sepharose, hydroxyapatite and 5´-AMP-Sepharose chromatography. The preparation appeared homogenous on SDS-PAGE. The enzyme is a tetrameric protein with a molecular weight of 200 kDa. A number of properties of the bovine brain enzyme, such as substrate specificity, molecular mass, kinetic parameters, and optimun pH have been determined and compared with those reported for preparations from other sources. The inhibition kinetic patterns obtained when the products of the reaction or substrate analogs are used as an inhibitor of the reaction catalyzed by the enzyme are consistent with an ordered sequential mechanism. The inhibition of succinic semialdehyde dehydrogenase by carbonyl compound o-phthalaldehyde was investigated. The inactivation followed pseudo-first order kinetics, and the second-order rate constant for the inactivation process was 25 M^(-1)S^(-1) at pH 8.4 and 25℃. The coenzyme NAD^(+) protected the enzyme against inactivation by o-phthalaldehyde, whereas the substrate succinic semialdehyde failed to prevent the reaction of o-phthalaldehyde with lysyl residues of the protein. The binding of approximately 3.7 mol or o-phthalaldehyde/mol of enzyme resulted in an irreversible loss of catalytic activity. The reaction was fast and easily monitored by absorption and fluorescence spectroscopy. These results suggest that the critical lysyl residues are located at or near the coenzyme binding site of the enzyme.
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