Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein.
Acetylene hydratase of the mesophilic fermenting bacterium Pelobacter acetylenicus catalyzes the hydration of acetylene to acetaldehyde. Growth of P. acetylenicus with acetylene and specific acetylene hydratase activity depended on tungstate or, to a lower degree, molybdate supply in the medium. The specific enzyme activity in cell extract was highest after growth in the presence of tungstate. Enzyme activity was stable even after prolonged storage of the cell extract or of the purified protein under air. However, enzyme activity could be measured only in the presence of a strong reducing agent such as titanium(III) citrate or dithionite. The enzyme was purified 240-fold by ammonium sulfate precipitation, anion-exchange chromatography, size exclusion chromatography, and a second anion-exchange chromatography step, with a yield of 36%. The protein was a monomer with an apparent molecular mass of 73 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was at pH 4.2. Per mol of enzyme, 4.8 mol of iron, 3.9 mol of acid-labile sulfur, and 0.4 mol of tungsten, but no molybdenum, were detected. The Km for acetylene as assayed in a coupled photometric test with yeast alcohol dehydrogenase and NADH was 14 microM, and the Vmax was 69 mumol.min-1.mg of protein-1. The optimum temperature for activity was 50 degrees C, and the apparent pH optimum was 6.0 to 6.5. The N-terminal amino acid sequence gave no indication of resemblance to any enzyme protein described so far.
Rosner
B M
BM
Fakultät für Biologie, Universität Konstanz, Constance, Germany.
Schink
B
B
D000079:Acetaldehyde / Q000378:metabolism; D000114:Acetylene / Q000378:metabolism*; D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D002845:Chromatography; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004227:Dithionite / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006836:Hydro-Lyases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017421:Sequence Analysis; D014025:Titanium / Q000494:pharmacology; D014414:Tungsten / Q000032:analysis*
D002951:Citrates; D007506:Iron-Sulfur Proteins; D004227:Dithionite; D019343:Citric Acid; D014025:Titanium; D006836:Hydro-Lyases; C096200:acetylene hydratase; D000079:Acetaldehyde; D000114:Acetylene; D014414:Tungsten