Purification and Some Properties of a Hitherto-Unknown Enzyme Reducing the Carbon-Carbon Double Bond of Alpha, Beta-Unsaturated Carboxylate Anions

Eur J Biochem. 1979 Jun;97(1):103-12. doi: 10.1111/j.1432-1033.1979.tb13090.x.

Abstract

2-Enoate-reductase, a previously unknown soluble enzyme is present in Clostridium kluyveri and another Clostridium species growing on (E)-2-butenoate. From the latter the reductase was purified 88-fold with an overall yield up to 74%. The enzyme was pure as judged by polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate as well as by isoelectric focusing. The purification of the enzyme was performed in the presence of (E)-2-methyl-2-butenoate as substrate to keep the enzyme in the oxidized state and under anaerobic conditions. The purification procedure included an ammonium sulphate precipitation, chromatography on DEAE-Sepharose CL-6B, hydroxylapatite and Sepharose CL-6B. The enzyme reduces different alpha,beta-unsaturated carboxylate anions such as (E)-2-butenoate, (E)-2-methyl-2-butenoate, (E)-cinnamate and probably many others in a NADH-dependent reaction to the saturated carboxylate anions. Fumarate, 3-phenyl-2-propinate, 2-enoyl-methyl and CoA esters proved not to be substrates for the purified reductase. NADPH does not act as an electron donor. The enzyme was shown to have a molecular weight of about 450,000 by gel chromatography. It consists of subunits with a molecular weight of 78,000. Per subunit about 1 FAD, 3.5--3.8 atoms of iron and 4.0 labile sulphur atoms have been found indicating a conjugated iron-sulphur flavo-protein. Copper could not be detected. The isoelectric point was 8.4. As shown by absorption spectroscopy the enzyme can be reduced by NADH and reoxidized with dichloroindophenol, hexacyanoferrate III, oxygen and substrates. Addition of 8 mol p-hydroxymercuribenzoate to 1 mol subunit completely destroyed the activity of the reductase. So far no physiological role of the enzyme is known.

MeSH terms

  • Amino Acids / analysis
  • Anaerobiosis
  • Clostridium / enzymology*
  • Fatty Acids, Unsaturated
  • Kinetics
  • Macromolecular Substances
  • Molecular Weight
  • Oxidoreductases / isolation & purification
  • Oxidoreductases / metabolism*
  • Spectrophotometry
  • Substrate Specificity

Substances

  • Amino Acids
  • Fatty Acids, Unsaturated
  • Macromolecular Substances
  • Oxidoreductases