Isotope Discrimination by Ribulose 1,5-Diphosphate Carboxylase

Abstract

Carbon 13 isotope di ntion by ribulose 1,5-diphosphate car-boxylase from soybean (Glycine max [Merr.J cv. Amsoy) was studied as a function of temperature, bicarbonate concentration, and pH. None of these factors affected the degree of discrimination against 13C. The average 813C was-28.3%., a value dose to that found for whole C3 plants. The zero temperature response observed here with ribulose 1,5-diphosphate carboxylase corroborates data from whole plants. The lack of effect of bicarbonate concentration on discrimination is consistent with both current theories of alternate forms of carboxylase. Terrestrial plants contain less 13C relative to '2C than is present in the CO2 in air (14). This discrimination probably occurs during the initial enzymic carboxylation of RuDP2 by RuDP carboxylase (EC 4.1.1.39) (18). Carbon dioxide has a small mol wt compared to other metabolites, and its atmospheric pool is large and incompletely utilized, unlike internal pools, and both these factors increase the possibility of observing isotopic effects. Whelan et al. (18) found that the 13C discrimination by RuDP carboxylase was dependent upon temperature. At 24 C the isotopic fractionation was-33.7%o, while at 37 C the fractionation was-18.3%o. Smith et al. (12) and Troughton and Card (14) found no effect of temperature on the carbon isotope ratio of whole C3 plants and this casts doubts on the role of the carboxyl-ase in 13C discrimination. It has recently been suggested that RuDP carboxylase exists in alternate states, dependent on the CO2 substrate concentration. Two hypotheses have been put forward to explain the observed kinetics: that the enzyme can exist in two forms with different Km(CO2) (1) and that the enzyme exists in either an inactive or an active form (8). As the Km hypothesis suggests that the enzyme may exist in two forms with different binding affinities for CO2 and because differences in binding energies between isotopes is one possible reason for discrimination (16), it is possible that the two enzyme states would discriminate against '3C-CO2 differently. On the other hand, active and inactive forms of the enzyme would not show differing isotope discrimination. Placing the enzyme in different activity states by running the reaction in different bicarbonate concentrations, and meas-Dedicated to the memory of J. W. Lyttleton. 2 Abbreviations: RuDP: ribulose 1 ,5-diphosphate; PGA: phospho-glyceric acid. uring the '3C/'2C ratio in the 3-P-glycerate formed would provide one test of the two theories. A state-dependent ratio would be positive proof against the active/inactive theory, while a constant ratio would be compatible with both theories. This paper reports the effect of temperature and HCO3-concentration on the discrimination against '3C-CO2 by purified soybean RuDP carboxylase. MATERIALS AND METHODS Materials. Ribulose 1,5-diphosphate was either purchased from Sigma Chemical Company or synthesized from Sigma ri-bose-5-P as described by Horecker et al. (6). Sodium bicarbon-ate was obtained from British drug houses. Purification of RuDP Carboxylase. Ribulose 1,5-diphosphate carboxylase was purified from either cabinet-or glasshouse-grown soybean (Glycine max [Merr.] cv. Amsoy) plants as described by Laing et al. (8) with the following modifications. The leaves were ground in 25 mm tris-Cl (pH 7.6), 10 mM MgCl2, and 20 mm NaHCO3 and only one 35 to 46% (NH4)2SO4 fractiona-tion step was used. Specific activities were between 0.6 and 1 ,umol/mg protein-min at 30 C and saturating substrates. ENZYME ASSAYS Temperature Series. The reactions were performed in four 10-ml test tubes closed with serum caps at each of four temperatures. Temperatures were controlled as described by Taylor et al. (13). The reaction mixtures for the temperature series contained 50 mM tris-Cl (pH 8), 10 mM MgCl2, 50 mM NaHCO3, 1 mM RuDP, and about 40 ,ug/ml of enzyme in a total volume of 28 ml. At this HCO3-concentration, RuDP oxygenase activity is negligible (2). Mixtures minus RuDP and enzyme were incubated for 30 min to ensure HCO3-/CO2 equilibrium. The RuDP was then added, and 1-ml aliquots were removed for determination of the 13C/'2C ratio of the HCO3-. Then the enzyme, activated by preincubation for 4 hr in 25 mm tris-Cl, (pH 8) and 10 mM MgCl2 at 0 C, was added, and the reactions were incubated for 30 min so that the RuDP was completely carboxylated. This ensured that no isotopic discrimination was contributed by selective carboxylation of RuDP. Subsequently another 1-ml HCO3-sample was taken, and the excess HCO3-was driven off with HCl03. The mixtures were then titrated to pH 6 with KOH and centrifuged to remove KC104 and denatured enzyme. The samples were applied to a Dowex column (Bio-Rad AG1 x8, 200-400 mesh) in the Cl-form and eluted stepwise with HCl. Purified 3-P-glycerate was eluted with 0.05 to 0.06 M HCl. A standard sample of 3-P-glycerate in the same reaction mix was treated in a similar manner to determine the discrimination caused by the purification procedure. 580