Ribulose biophosphate carboxylase from Thiobacillus A2. Its purification and properties.
Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an Srel. of 16.9. Km for RuBP was 122 muM, for total "CO2" it was 4.17 mM, and for Mg2+ 20.0 muM. The absolute requirement for a divalent cation was satisfied by Mg2+ which was replaceable to a certain extent by Mn2+. Activity was not significantly affected by SO(2-4), SO(2-3), or S(2)O(2-3) at 1.0 mM. At this concentration S(2-) caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.04 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a Ki of 20 muM. Lost activity could be restored partially by GSH or Cu2+. Although azide at the concentration tested had no significant effect on enzyme activity, 2, 4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.
D000227:Adenine Nucleotides / Q000494:pharmacology; D002245:Carbon Dioxide / Q000378:metabolism; D002262:Carboxy-Lyases / Q000032:analysis*; D003486:Cyanides / Q000494:pharmacology; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008970:Molecular Weight; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013855:Thiobacillus / Q000201:enzymology*; D014461:Ultracentrifugation
D000227:Adenine Nucleotides; D003486:Cyanides; D007477:Ions; D013439:Sulfhydryl Reagents; D002245:Carbon Dioxide; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase