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. Gro wth of P. acetylenicus with acetylene and specific acetylene hydratase activity depended on tungstate or, to a lower degree, molybdate suppl y in the medium, The specific enzyme activity in cell extract was high est after growth in the presence of tungstate, Enzyme activity was sta ble even after prolonged storage of the cell extract or of the purifie d protein under air, However, enzyme activity could be measured only i n the presence of a strong reducing agent such as titanium(III) citrat e or dithionite, The enzyme was purified 240-fold by ammonium sulfate precipitation, anion exchange chromatography, size exclusion chromatog raphy, and a second anion-exchange chromatography step, with a yield o f 36%, The protein was a monomer with an apparent molecular mass of 73 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel elect rophoresis, 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 K-m for acetylene as assayed in a coupled photometric test with yeast alcohol dehydrogenase and NADH was 14 mu M, and the V-max was 69 mu mol . min-1 . mg of protein(-1), The optimum temperature for activity was 50 degrees C, and the apparen t pH optimum was 6.0 to 6.5, The N-terminal amino acid sequence gave n o indication of resemblance to any enzyme protein described so far.