Properties and regulation of ribulose diphosphate carboxylase from Thiobacillus novellus.
Ribulose-diphosphate carboxylase from Thiobacillus novellus has been purified to hemogeneity as observed by polyacrylamide gel electrophoresis and U.V. light observation during sedimentation velocity analysis. The optimum pH for the enzyme with Tris-HCl buffers was about 8.2. Concentrations of this buffer in excess of 80 mM were inhibitory. The apparent Km for RuDP was about 14.8 muM with a Hill value of 1.5, for HCO3- the apparent Km was about 11.7 mM with an n value of 1.18 and for Mg2+ about 0.61 mM. The enzyme was specific for this cation. Relatively high concentrations of either Hg2+ or pCMB were required before significant inhibition was observed. Activity declined slowly during a 4-hr incubation period in either 3.0 M or 8.0 M urea. Incubation for 12 hrs resulted in complete loss of activity which was not prevented by 10 mM Mg2+ and was not reversed by dialysis and subsequent addition of 10 mM cysteine. Polyacrylamide gel electrophoresis revealed a loss of the major band and the appearance of 2 new bands. SDS polyacrylamide gel electrophoresis gave an average M.W. of 73500 +/- 2500 for the slower moving band and 12250 +/- 2500 for the faster moving. However, incubation in urea for up to 40 hrs revealed a decrease in the M.W. of the slower moving band to about 60000. The Ea for the enzyme was calculated to be about 18.85 kcal mole-1, with the possibility of a "break" between 40 and 50 degrees C. The Q10 was 3.07 between 20 and 30 degrees C whereas between 30 to 40 degrees C it was 3.31. Only phosphorylated compounds caused significant inhibition of enzyme activity. They included ADP, FDP, F6P, G6P, PEP, 6PG, 2-PGA, R1P, R5P, and Ru5p.
D002245:Carbon Dioxide / Q000378:metabolism; D002262:Carboxy-Lyases / Q000032:analysis*; D002413:Cations, Divalent; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004790:Enzyme Induction; D004794:Enzyme Repression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D013403:Sugar Phosphates / Q000494:pharmacology; D013855:Thiobacillus / Q000201:enzymology*; D014508:Urea / Q000494:pharmacology
D002413:Cations, Divalent; D002729:Chloromercuribenzoates; D013403:Sugar Phosphates; D002245:Carbon Dioxide; D014508:Urea; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008628:Mercury