Characterization of the recombinant succinic semi‐aldehyde dehydrogenase from Saccharomyces cerevisiae |
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Authors: | Juxiang Cao Narendra K Singh Robert D Locy |
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Affiliation: | 1. Department of Biological Sciences, Auburn University, , AL, USA;2. Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, , Boston, MA, USA |
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Abstract: | The yeast succinic semi‐aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS–PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µm NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi‐aldehyde (Km = 15.48 ± 0.14 µm ) and could use both NAD+ and NADP+ as co‐factors, with Km values of 579.06 ± 30.1 µm and 1.017 ± 0.46 mm, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD+ (Ki = 129.5 µm ) but a non‐competitive inhibitor with respect to succinate semi‐aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with Ki = 1.13–10.2 mm, and showed competitive inhibition with respect to NAD+ and mixed‐competitive, non‐competitive and non‐competitive inhibition, respectively, with respect to succinate semi‐aldehyde. The kinetic data suggest a 'ping‐pong' mechanism. Copyright © 2014 John Wiley & Sons, Ltd. |
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Keywords: | Saccharomyces succinate semi‐aldehyde dehydrogenase SSADH protein purification |
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