Ryan F J, Tolbert N E
J Biol Chem. 1975 Jun 10;250(11):4229-33.
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.
核酮糖二磷酸羧化酶和加氧酶活性特性的相似性进一步证实了同一蛋白质催化这两种反应的假说。用氧电极在空气中测定时，核酮糖二磷酸加氧酶的Km（核酮糖二磷酸）为0.33 mM。在10至35 mM MgCl2存在下可获得最大活性。较高浓度的MgCl2具有抑制作用，但会使最适pH从9.3或9.4变为8.7或9.0。MnCl2是加氧酶的有效辅因子，CoCl2存在时也有一定活性。菠菜叶纯化蛋白的核酮糖二磷酸羧化酶和加氧酶活性在0℃储存时会缓慢失活，若同时存在25 mM MgCl2和1 mM二硫苏糖醇，则在50℃下10分钟可重新激活。该酶的巯基与5, , dithiobis(2 - nitrobenzoic acid)快速反应，在pH 7.8时约为4个，在pH 9.4时约为11个。在这两个pH值下，核酮糖二磷酸可阻止其中两个巯基与该试剂反应。在pH 9.0时，3 mM核酮糖二磷酸存在下50 mM碳酸氢盐会使加氧酶活性受到约50%的抑制，根据这些参数的变化，这种抑制作用归因于CO2种类。丙烯酰胺凝胶中的纯化酶在超氧自由基存在下可阻止硝基蓝四唑的还原，但溶液中的酶不具有超氧化物歧化酶的反应活性。
