title: Characterization of the recombinant succinic semi-aldehyde dehydrogenase from Saccharomyces cerevisiae.
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.
authors.affiliation: Department of Biological Sciences, Auburn University, AL, USA; Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Juxiang
authors.initials: J
authors.lastname: Cao
authors.forename: Narendra K
authors.initials: NK
authors.lastname: Singh
authors.forename: Robert D
authors.initials: RD
authors.lastname: Locy
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism
chemical_list: D000447:Aldehydes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D019802:Succinic Acid; D000444:Aldehyde Dehydrogenase; D013385:Succinate Dehydrogenase
keywords: SSADH; Saccharomyces; protein purification; succinate semi-aldehyde dehydrogenase