Characterization of the recombinant succinic semi‐aldehyde dehydrogenase from Saccharomyces cerevisiae. Issue 10 (8th September 2014)

• Record Type: Journal Article
• Authors: Cao, Juxiang; Singh, Narendra K.; Locy, Robert D.
• Is Part Of: Yeast. Volume 31:Issue 10(2014:Oct.)
• Page Start: 411
• Page End: 420
• Publication Date: 2014-09-08
• Subjects: Yeast -- Periodicals; Yeasts -- Periodicals; Yeasts -- genetics -- Periodicals; Electronic journals; 547
• DOI: 10.1002/yea.3035
• Languages: English
• ISSNs: 0749-503X

Abstract

<abstract abstract-type="main"> <title>Abstract</title> <p>The yeast succinic semi‐aldehyde dehydrogenase gene (<italic>SSADH</italic>; EC 1.2.1.16) was cloned and overexpressed in <italic>Escherichia coli</italic>. 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 µ<sc>m</sc> NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi‐aldehyde (<italic>K</italic>_m = 15.48 ± 0.14 µ<sc>m</sc>) and could use both NAD⁺ and NADP⁺ as co‐factors, with <italic>K</italic>_m values of 579.06 ± 30.1 µ<sc>m</sc> and 1.017 ± 0.46 m<sc>m, </sc> respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD⁺ (<italic>K</italic>_i = 129.5 µ<sc>m</sc>) but a non‐competitive inhibitor with respect to succinate semi‐aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with <italic>K</italic>_i = 1.13–10.2 m<sc>m, </sc> 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 &amp; Sons, Ltd.</p> </abstract>