title: E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond.
abstract: The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.
authors: Department of Genetics, Development, and Cell Biology, Center for Biorenewable Chemicals, Iowa State University, Ames, IA 50010, USA. yimingguo@yahoo.com
authors: Y
authors: Y
authors: Guo
authors: D J
authors: DJ
authors: Oliver
mesh_terms: D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases; C068072:propionate - CoA ligase; D003545:Cysteine; D004229:Dithiothreitol