Ribulose 1,5-bisphosphate carboxylase/oxygenase from Pseudomonas oxalacticus.
Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified by a rapid, facile procedure from formate-grown Pseudomonas oxalaticus. The electrophoretically homogeneous enzyme had specific activities of 1.9 mumol of CO2 fixed per min per mg of protein and 0.15 mumol of O2 consumed per min per mg of protein. The amino acid composition was similar to that of other bacterial sources of the enzyme. The molecular weights determined by sedimentation equilibrium and by gel filtration were 421,000 and 450,000, respectively. Upon sodium dodecyl sulfate electrophoresis of enzyme purified under conditions which would limit proteolysis, two types of large (L) subunits and two types of small (S) subunits were observed with apparent molecular weights of 57,000, 55,000, 17,000 and 15,000. By densitometric scans at two different protein concentrations the stoichiometry of the total large to total small subunits was 1:1, implying an L6S6 structure. Electron micrographs of the enzyme revealed an unusual structure that was inconsistent with a cubical structure. The enzyme had an unusually high Km for ribulose 1,5-bisphosphate (220 microM) and was strongly inhibited by 6-phosphogluconate in the ribulose 1,5-bisphosphate carboxylase assay (Ki = 270 microM). One, 5, and 12 days after purification the enzyme was half-maximally activated at 0.13 microM, 0.23 mM, and 0.70 mM CO2, respectively, at saturating Mg2+. At saturating CO2, enzyme 1 day afer purification responded sigmoidally to Mg2+ and was half-maximally activated by 0.85 mM Mg2+ in the absence of 6-phosphogluconate (Hill coefficient, h = 2.0) and by 0.19 mM Mg2+ in the presence of mM 6-phosphogluconate (h = 1.7).
D000596:Amino Acids / Q000032:analysis; D002245:Carbon Dioxide / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000378:metabolism*; D004789:Enzyme Activation; D005942:Gluconates / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008854:Microscopy, Electron; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D012274:Ribulosephosphates / Q000378:metabolism
D000596:Amino Acids; D005942:Gluconates; D012274:Ribulosephosphates; D002245:Carbon Dioxide; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium