for the Existence of Discrete Activator and Substrate Sites for CO, on Ribulose-1,5-bisphosphate Carboxylase*

When incubated with CO2 and Mg2+, ribulose-1,5-bis-phosphate carboxylase forms a ternary complex of enzyme . CO2 . Mg. This complex was prepared using high specific activity [14C]O2 and injected into a solution containing a large (50- to 112-fold) molar excess of [12C]O2 and sufficient ribulose 1,5-bisphosphate to permit the catalytic site to turn over several times. The enzyme was then rapidly separated from the other components by gel filtration and its radiospecific activity was determined to be 30 to 60 times that of the medium. If the CO2 activator and the CO2 substrate sites were one and the same, then, following turnover, the enzyme should have been in isotopic equilibrium with the medium. The finding that this was not the case, by a factor of about 40, indicates that the CO2 activator site is physically distinct from the CO2 substrate site.


When incubated
with CO2 and Mg"', ribulose-l,&bisphosphate carboxylase forms a ternary complex of enzyme l COZ. Mg. This complex was prepared using high specific activity ['"C]O, and injected into a solution containing a large (SO-to 112-fold) molar excess of ["C]02 and sufficient ribulose l,&bisphosphate to permit the catalytic site to turn over several times. The enzyme was then rapidly separated from the other components by gel filtration and its radiospecific activity was determined to be 30 to 60 times that of the medium.
If the CO2 activator and the COZ substrate sites were one and the same, then, following turnover, the enzyme should have been in isotopic equilibrium with the medium. The finding that this was not the case, by a factor of about 40, indicates that the COZ activator site is physically distinct from the COZ substrate site.
The activity of ribulose-bisphosphate carboxylase (EC 4.1.1.39) has long been known to be stimulated by preincubation with COZ' and Mg2+ (3). Kinetic studies indicate that the activation of the enzyme is associated with the ordered formation of a ternary enzyme. COZ. Mg complex (1,2,4,5 a 55-fold molar excess of ["C]O2 and sufficient RuBP to permit an average of 10 turnovers. Thus, sufficient enzyme was present to drive the reaction to completion within about 8 s. Acid-stable 14C radioactivity, determined on aliquots withdrawn after mixing, was constant from the earliest sampling time, 20 s, confirming that the reaction proceeded rapidly to completion. The resultant mixture was rapidly transferred to a small gel filtration column equilibrated with 0.1 M Tris-HCl, 0.1 M MgC12, 0.01 M NaHC03, pH 9.02, and elution was performed with the same buffer. The result of such a procedure is shown in Fig. 1 acid-labile, alkali-stable "'C radioactivity; A-A, 14C radiospecific activity of enzyme-bound COZ. RuBP carboxylase ((6.3 mg); 90 nmol of enzyme protomer) was preincubated with 45.5 mM MgCL, 7.82 mM NaH['4C]Oo (8640 dpm/ nmol), 20 mM N,W-bis(2-hydroxyethyl)glycine (Bicine) NaOH, pH 8.2, in a total volume of 220 ~1 for about 15 min at 25°C. Then 200 pl of the above solution were injected into 795 ~1 of a rapidly stirred solution containing 31.4 mM MgCl,, 1.04 mM RuBP (sufficient for 10 turnovers/protomer site), 108 mM Hepes, 108 mM NaHCO:I, pH 8.35. After 20 and 40 s, 50-~1 aliquots were withdrawn to determine the quantity of acid-stable 14C! radioactivity. Seven hundred and fifty microliters of the remainder was immediately applied to a column (10 x 180 mm) of Bio-Gel P-4 equilibrated at 4°C with 0.1 M Tris-HCl, 0.1 M MgC12, 0.010 M NaHC03, pH 9.02, and eluted with the same buffer. One-half-milliliter fractions were collected. Protein was determined at 280 nm (5). Acid-stable 14C radioactivity was determined by mixing a 100~~1 aliquot with 250 pl of 10% (v/v) formic acid, taking the mixture to dryness and counting by scintillation spectrometry. Acid-labile, alkali-stable "'C radioactivity was determined by mixing a 100~pl aliquot with 100 ~1 of 10% (v/v) ethanolamine in ethanol and counting by scintillation spectrometry.
when Mg'+ was omitted from the elution buffer (data not shown).
The recovery of enzyme-bound 14C was in the range of 20 to 75% (Table I), depending upon the temperature at which the gel filtration step was performed. Increasing the temperature brought about greater dissociation of the enzyme. ['4C]02. Mg complex. The recovery of this complex was not 100%. Some dissociation of the complex or exchange with the [12C]Oz present in the elution buffer was to be anticipated during chromatography.
Although not apparent in Fig. 1 (for reasons of scale), the acid-labile, alkali-stable 14C radioactivity did not quite decline to zero between the two major peaks. This indicates, as one would expect, that the enzyme. ['4C]02. Mg complex has undergone partial dissociation during the period of chromatography thus causing this trailing phenomenon. Nevertheless, the specific radioactivity within the four fractions encompassing the protein peak (Fractions 9 to 12) was constant at about 4700 dpm/nmol of enzyme protomer. This value is 30 times the specitic radioactivity of the COZ in the medium after mixing. A summary of the results of six such experiments is presented in Table I.
The radiospecific activity of the product, 3-phosphoglycerate (corrected for the formation of 2 mol of product/m01 of ['4C]02 fiied) was consistently larger than the radiospecific activity of the COz in the medium after mixing. This result implies that a small quantity of the high specific activity ['4C]02, presumed to be present at the substrate CO2 site before mixing, was subsequently trapped with RuBP before it underwent exchange with ["C]02.

DISCUSSION
The strategy underlying the experiments reported here depends upon bringing the CO2 at the substrate site into isotopic equilibrium with the unbound CO* by permitting limited turnover to occur. By allowing an average of 3 to 25 turnovers/site, the possibility of "misses" occurring (a site failing to turnover) should have been minimized. It, therefore, seems reasonable to assume that aU of the substrate CO2 sites have turned over at least once. If, then, the activator CO2 and the substrate COZ are one and the same, it follows that the radiospecific activity of the enzyme should equal that of the unbound COz. But the observed radiospecific activity of the enzyme was some 40 times that of the unbound Con, even after having been subject to gel filtration against a buffer containing 10 mM NaHC03 (about 20 PM CO2 at pH 9.02). This result can only have occurred if the activator CO2 and substrate CO2 sites are physically distinct.
It is clear from the results reported here and elsewhere (1, 5, 9) that Mg2+ in some way stabilizes the activator CO2 molecule. It has been suggested that the activation of the enzyme involves the formation of a carbamate, which would provide a binding site for divalent cation and enhance the stability of the carbamate (1)  n Each of the six experiments was performed essentially as outlined in the legend to Fig. 1. However, different quantities of enzyme or RuBP were used in each case thus giving rise to different values for the number of turnovers/enzyme site and to the 14C radiospecific activity of the CO* after mixing.
b Refers to the temperature at which the gel filtration step was performed.
' The number of turnovers/site was computed from the molar ratio of RuBP to enzyme protomer. Enzyme protomer is defined as the 70,000-dalton species containing one large subunit and one small subunit.
d The 14C radiospecific activity of the enzyme-bound CO* is expressed as dpm/nmol of enzyme protomer. e Fixed CO* was determined from the quantity of acid-stable 14C radioactivity remaining following the reaction and from the quantity of RuBP supplied.
'The values in parentheses refer to the yield of enzyme-bound 14C radioactivity recovered after gel ftitration, assuming that 8640 dpm/ mnol represents 100%.