Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.
Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.
D001639:Bicarbonates / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D003035:Cobalt / Q000494:pharmacology; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D012274:Ribulosephosphates; D013696:Temperature; D013997:Time Factors
D001639:Bicarbonates; D012274:Ribulosephosphates; D003035:Cobalt; D008345:Manganese; D010105:Oxygenases; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium