(Received for publication, September 4, 1996, and in revised form, December 12, 1996)
From the Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, Australian Capital Territory 2601, Australia
The large subunit core of ribulose-bisphosphate
carboxylase from Synechococcus PCC 6301 expressed in
Escherichia coli in the absence of its small subunits
retains a trace of carboxylase activity (about 1% of the
kcat of the holoenzyme) (Andrews, T. J (1988) J. Biol. Chem. 263, 12213-12219). During steady-state
catalysis at substrate saturation, this residual activity diverted
approximately 10% of the reaction flux to
1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate as a result of elimination of inorganic phosphate from the first reaction intermediate, the 2,3-enediol form of ribulose bisphosphate. This indicates that the active site's ability to stabilize and/or retain this intermediate is compromised by the absence of small subunits. Epimerization and isomerization of the substrate resulting from misprotonation of the enediol intermediate were not significantly exacerbated by lack of small subunits. The residual carboxylating activity partitioned product between pyruvate and 3-phosphoglycerate in
a ratio similar to that of the holoenzyme, indicating that stablization
of the penultimate three-carbon aci-acid intermediate is
not perturbed by lack of small subunits. The underlying instability of
the five-carbon enediol intermediate was revealed, even with the
holoenzyme, under conditions designed to lead to exhaustion of
substrate CO2 (and O2). When carboxylation (and
oxygenation) stalled upon exhaustion of gaseous substrate, both spinach
and Synechococcus holoenzymes continued slowly to
eliminate inorganic phosphate from and to misprotonate the enediol
intermediate. With carboxylation and oxygenation blocked, the products
of these side reactions of the enediol intermediate accumulated to
readily detectable levels, illustrating the difficulties attendant upon
ribulose-P2 carboxylase's use of this reactive species as
a catalytic intermediate.
The photosynthetic enzyme Rubisco1 (EC
4.1.1.39) catalyzes the addition of CO2 to
ribulose-P2, producing two molecules of P-glycerate using a
multistep reaction sequence involving at least three enzyme-bound
catalytic intermediates (Scheme 1) (for reviews, see
Refs. 1-3). Two of these intermediates, the first and the third, are
strong nucleophiles by virtue of their enediol character. Both are
susceptible to side reactions that abort the carboxylation sequence and
compromise the catalytic efficiency of the enzyme.
The first of these intermediates is the 2,3-enediol form of
ribulose-P2 produced by removal, by an enzymatic base, of
the proton attached to C-3 of ribulose-P2. There may be two
or more differently protonated forms of this intermediate; only the
enediol form is shown in Scheme 1. Collectively, we refer to all forms of this intermediate as "the enediol." This is the species that must be attacked by CO2 at C-2 and (concertedly or
sequentially) by H2O at C-3 to form the six-carbon
intermediate and thus allow carboxylation to proceed (Scheme 1). Three
different classes of side reactions are known for this intermediate.
First, the enediol is also attacked by O2 at C-2, resulting
in the formation of 2-phosphoglycolate which is, in turn, partially
recycled to photosynthetic metabolism by the photorespiratory glycolate
pathway (4-6). Second, the enediol can be reprotonated incorrectly,
producing pentulose bisphosphate isomers of the substrate
(xylulose-P2 and 3-ketoarabinitol-P2) (7-12)
which are strong inhibitors and/or very weak substrates of the enzyme
(13-15). Third, mutants of Synechococcus PCC 6301 and
Rhodospirillum rubrum Rubiscos were discovered that were
compromised in their ability to stabilize the enediol. These mutant
enzymes catalyzed the production of varying amounts of
deoxypentodiulose-P as a result of elimination of the phosphoryl
group attached to C-1 of the intermediate (P1) (16, 17).
The final intermediate in the carboxylation sequence is produced
following cleavage of the C-2/C-3 bond of the six-carbon intermediate
(Scheme 1). This aci-acid form of the P-glycerate molecule,
derived from carbons 1 and 2 of ribulose-P2 and the incoming CO2 molecule, requires stereospecific protonation
at C-2 to form P-glycerate. Only a single kind of side reaction is known for this intermediate, elimination of the phosphoryl moiety to produce enol-pyruvate and thus pyruvate (18). All
wild-type Rubiscos studied so far partition approximately 0.7% of
their aci-acid intermediate to pyruvate (18), and mutants
are known in which this partitioning ratio is increased or decreased
(14, 16, 17). Thus both of Rubisco's enediol-like intermediates are
prone to
elimination of the P1 phosphate group, and there may be a
common reason (16, 18). Stereoelectronic considerations suggest that
the
elimination tendency is least when the
substituent is
maintained coplanar with the enediol double bond. However, a single
disposition of the C-1/bridge O bond cannot be found which satisfies
this requirement for both intermediates because the double bond is
differently positioned in the two intermediates. In order to maintain
co-planarity with the double bond, the C-1/bridge-O bond must change
its dihedral angle with respect to the C-2/carboxyl-C bond of the
six-carbon and aci-acid intermediates through 90° following hydrolysis of the six-carbon intermediate (Scheme 1). This
movement may not occur rapidly or completely enough to stabilize the
aci-acid intermediate fully.
The ability to produce a small subunit-free version of
Synechococcus Rubisco by expression of the cyanobacterial
rbcL gene in Escherichia coli without the
companion rbcS gene presents an opportunity for study of the
influence of the small subunits on the catalytic mechanism. The large
subunits assemble correctly into an octameric core that retains
approximately 1% of the holoenzyme's kcat but
with weaker substrate affinities (19). Although it is known that the
L8 form distinguishes between CO2 and
O2 as substrates similar to the
L8S8 holoenzyme (20), information about the
ability of L8 to suppress other side reactions is lacking. Such information would contribute toward understanding the way the
small subunits influence the active-site structure to increase its
activity and alter its kinetic properties. Because small subunits do
not bind near the active site and make no direct contribution to its
structure (21), such influences must be effected through indirect
conformational changes. If the small subunit alters the way the P1
phosphate group is bound, as has been speculated from crystallographic
structural information (22), then absence of the small subunit might be
expected to perturb the ability to suppress elimination side
reactions.
In this study, we have assessed the extent of side reactions of the
carboxylation sequence catalyzed by wild-type Rubiscos. We have avoided
complications associated with the competing oxygenation pathway, itself
prone to side reactions at least with mutant Rubiscos (23, 24), by
excluding O2. In the case of the Synechococcus enzyme, we have studied both the L8S8
holoenzyme and its L8 core without small subunits. To
amplify accumulation of side products derived from the enediol
intermediate with both Synechococcus and spinach
holoenzymes, we have used the device of allowing exhaustion of gaseous
substrates to occur while a saturating concentration of
ribulose-P2 still remains. Our data show that, even with
wild-type Rubiscos, elimination and misprotonation of the enediol
intermediate are easily detectable and that
elimination of the
enediol, but not other side reactions, is strikingly exacerbated by the
absence of small subunits.
Ribulose-P2 (25), [1-14C]ribulose-P2 (26), and [carboxyl-14C]carboxypentitol-P2 (27) were prepared as described. NaH14CO3 was obtained from Amersham Corp. and o-phenylenediamine from Sigma. Rubisco holoenzyme (L8S8) expressed in E. coli from the rbcL and rbcS genes of Synechococcus PCC 6301 was purified as described (16). Spinach Rubisco was purified using a procedure involving polyethylene glycol precipitation followed by anion-exchange chromatography on a Waters Protein-Pak Q column, essentially as described by Edmondson et al. (7) but omitting the final gel filtration step.
Preparation of L8 RubiscoE. coli HB101 harboring plasmid pDB53, which bears the Synechococcus PCC 6301 rbcL gene under the transcriptional control of the lac promoter (28), was used to produce large subunit octamers (L8) devoid of small subunits. L8 was extracted from E. coli, purified, and stored using the same procedures developed for the Synechococcus L8S8 holoenzyme (16). Immediately before use, L8 preparations were dialyzed overnight at 4 °C against 20 mM potassium phosphate buffer, pH 7.6, containing 1 mM dithiothreitol and concentrated using a Centricon 30 unit (Amicon).
Carboxylase AssayThe carboxylase activity of L8 Rubisco was routinely measured by 14CO2 fixation both in the presence and in the absence of added small subunits (19, 29).
Measurement of Pyruvate ProductionPyruvate was determined
by measuring [14C]lactate formed during complete
consumption of ribulose-P2 by Rubisco in the presence of
14CO2, lactate dehydrogenase, and NADH. The
assay solution contained purified Synechococcus Rubisco
(final concentrations, 150 µg·ml1 of L8
or 5 µg·ml
1 of L8S8), 100 mM Epps-NaOH buffer, pH 8.0, 20 mM
MgCl2, 0.1 mg·ml
1 of bovine serum albumin,
and 20 mM NaH14CO3 (30 Bq·nmol
1). After preincubation for 10 min at 25 °C,
4 units·ml
1 of rabbit muscle lactate dehydrogenase
(Boehringer Mannheim) and 0.2 mM NADH were added, and
catalysis was initiated by adding 2 mM
ribulose-P2. The assay solution was then incubated at
25 °C for a further 16 h. Unreacted
NaH14CO3 was removed by adding
H3PO4 to reduce the pH to 3.0 and vortexing for
5 min. Enzymes were then removed by filtration through Millipore Ultrafree-MC filters (10,000 nominal Mr
cut-off), and a sample of the filtrate was chromatographed on a 5-µm
Nucleosil C18 reverse-phase column (Macherey-Nagel,
0.46 × 25 cm) eluted at 0.9 ml·min
1 with 0.1%
(v/v) aqueous trifluoroacetic acid (Merck, spectroscopic grade), pH
1.6, as the mobile phase. Fractions of 0.1 ml were collected and mixed
with 3 ml of scintillant for scintillation counting. Pyruvate
production was calculated as the ratio of 14C in lactic
acid to the total 14C in lactic, glyceric, and
3-phosphoglyceric acids.
Experiments were
conducted with Synechococcus L8S8
and L8 under anaerobic and CO2-saturating
conditions. The enzymes were placed in the main compartment of a
Warburg flask in 2 (L8S8) or 0.5 (L8) ml (final volume) of a solution containing 50 mM triethanolamine acetic acid buffer, pH 8.3, 20 (L8S8) or 8 (L8) mM
magnesium acetate, and 40 mM NaHCO3. The
L8 reaction also contained 10 mM
K2HPO4 and 1 mM dithiothreitol. The
phosphate was necessary to maintain solubility of L8 and
its presence also necessitated the use of a lower Mg2+
concentration to avoid precipitation. Controls lacking enzyme and with
decarbamylated L8S8 (minus Mg2+,
plus 1 mM EDTA) were also included using otherwise the same conditions as for L8S8. The side arm of the
flasks contained 0.59 µmol of
[1-14C]ribulose-P2 (5.9 Bq·nmol1). The flasks were installed in a Warburg
apparatus, and an atmosphere of humidified 1% (v/v) CO2 in
N2 was passed continuously through the flask during all
stages of the experiment. After pre-equilibration with shaking for 30 min at 25 °C, the contents of the side arm were mixed with those of
the main compartment to initiate the reaction. Shaking was stopped
20 s afterward, and incubation was continued for 2 h. To
terminate the reaction, 0.1 volume of 0.5 M
NaBH4 in 0.1 N NaOH (equilibrated under
N2) was added via a septum-capped injection port, and the
mixture was shaken for a further 30 min. Borohydride was discharged
with Bio-Rad AG 50W-X8 resin (H+ form), and the sample was
prepared for anion-exchange chromatography as described (16). A sample
was applied to a 5-µm Spherisorb SAX column (0.46 × 25 cm,
Alltech) and eluted with a phosphate gradient as described previously
(16). Fractions comprising individual radioactive peaks were pooled,
incubated with alkaline phosphatase, passed through Bio-Rad AG 1-X8
(formate) resin, and chromatographed on an Aminex HPX-87C column as
described previously (16).
Additional experiments were conducted with Rubisco holoenzymes from
spinach and Synechococcus under conditions designed to lead
to exhaustion of CO2/HCO3
(and any traces of O2) while still leaving an excess of
unreacted ribulose-P2. In this case, the enzymes were
placed in the main compartment of the Warburg flask in 1 (spinach) or
0.5 (Synechococcus) ml (final volume) of a solution
containing 92 mM triethanolamine acetic acid buffer, pH 8.3 (spinach) or 7.8 (Synechococcus), 23 mM
magnesium acetate, 0.7 mM EDTA, 4 (spinach) or 3 (Synechococcus) mM NaHCO3, and 0.5 mg·ml
1 bovine carbonic anhydrase. Controls containing,
in addition, 3 mM carboxypentitol-P2 were also
included for each enzyme. The side arms of the flasks contained
[1-14C]ribulose-P2 (6.4 (spinach) and 3.2 (Synechococcus) µmol (4 Bq·nmol
1)). The
reaction mixtures were shaken for 15 min at 25 °C in a Warburg
apparatus while an atmosphere of humidified 0.1% (v/v) CO2
in N2 was passed through. The flasks were then sealed
before the contents of the side arm were mixed with those of the main compartment to initiate the reaction. Shaking was stopped 30 s later, and incubation was continued for 1 (spinach) or 3 (Synechococcus) h before termination. Termination and
subsequent procedures were the same as described above for the
saturating CO2 experiments.
For both CO2-saturating and CO2-limiting sets
of experiments, the amounts of the different pentulose bisphosphates
present at quenching were calculated from the amounts of radioactivity present in ribitol, arabinitol, and xylitol determined by
chromatography on the Aminex HPX-87C column. The calculation assumes
that the only bisphosphates present before reduction were those of
ribulose, xylulose, and 3-ketoarabinitol, that reduction of
ribulose-P2 produced the ribitol and arabinitol isomers in
the same ratio as that observed in the minus-enzyme or
plus-carboxypentitol-P2 controls, and that reduction of
xylulose-P2 produced the arabinitol and xylitol isomers in
equal amounts. Thus, [ribulose-P2] = (1 + )R; [xylulose-P2] = 2X; and
[3-ketoarabinitol-P2] = A
R
X, where R, A, and
X are the concentrations of the bisphosphates of ribitol,
arabinitol, and xylitol, respectively, present at quenching, and
is
the arabinitol/ribitol ratio observed in the minus-enzyme or
plus-carboxypentitol-P2 controls. The value of
averaged
0.76 in the present experiments.
Deoxypentodiulose-P produced during catalysis by
wild-type spinach and Synechococcus Rubiscos was measured
continuously by exploiting the absorbance at 330 nm of the quinoxaline
adduct with o-phenylenediamine. Reaction mixtures contained,
in a total volume of 2 ml, 60 mM Epps-NaOH buffer, pH 7.9, 12 mM MgCl2, approximately 3 mM
NaHCO3, 0.6 mM EDTA, 20 mM
o-phenylenediamine, 0.5 mg·ml1 of bovine
carbonic anhydrase, and 3 to 4 mg·ml
1 of Rubisco. A
control, lacking Rubisco, was also included. All components except the
Rubisco were dissolved in N2-sparged solutions. The
reaction mixture was placed in a septum-capped spectrophotometer cuvette, and the headspace was flushed with N2. After
several minutes pre-equilibration at 25 °C, the reaction was
initiated at time 0 by adding ribulose-P2 solution from a
syringe to bring the final concentration to 6.1 mM. The
ribulose-P2 solution had been neutralized to pH 6.8 and
sparged with N2 just before use. The absorbance was
monitored at 330 nm, and 20-µl aliquots were withdrawn with a syringe
at intervals and added to 500 µl of 2% (w/v) perchloric acid at
4 °C. These samples were neutralized with
K2CO3 and centrifuged, and aliquots of the
supernatant were assayed for P-glycerate spectrophotometrically in a
2-ml assay solution containing 92 mM Epps-NaOH, pH 8.0, 18 mM MgCl2, 0.2 mM NADH, 50 mM phosphocreatine, 12 units of rabbit muscle creatine phosphokinase, 36 units of yeast P-glycerate kinase, 15 units of rabbit
muscle glyceraldehyde-phosphate dehydrogenase, 36 units of rabbit
muscle triose-phosphate isomerase, and 12 units of rabbit muscle
glycerol-phosphate dehydrogenase (all enzymes from Boehringer Mannheim). P-glycerate content was measured from the reduction in
absorbance at 340 nm which followed addition of ATP to 50 µM. Experiments with deoxypentodiulose-P produced by
prolonged room temperature storage of ribulose-P2 at pH 8 (30) showed that the quinoxaline adduct is formed with a half-time of
approximately 60 s under these assay conditions. Therefore,
although adduct formation does induce a lag in these assays, it is not
an unacceptably long one, given the slow rate of deoxypentodiulose-P
formation and the long assay periods used. The amounts of
deoxypentodiulose-P produced were calculated using the molar
absorptivity of the quinoxaline adduct determined by measuring
inorganic phosphate (31) released from chromatographically isolated
adduct by alkaline phosphatase. The value obtained (9300 M
1·cm
1 in aqueous solution at
pH 8) approximates those reported for other quinoxalines (32).
Protein was determined with the
Cu-bicinchoninic acid procedure (33) using Pierce reagents or by
measuring the absorbance at 280 nm (34, 35). L8
concentrations were calculated from determinations of its carboxylase
activity, assuming a specific activity of 50 nmol·min1·mg
1 under the standard assay
conditions where the bicarbonate concentration (25 mM) is
approximately half-saturating for L8 (19).
The partitioning of
Rubisco's aci-carbanion intermediate between its
alternative products, P-glycerate and pyruvate (measured as lactate),
was determined for both the L8 core and the
L8S8 holoenzyme forms of
Synechococcus Rubisco. In this study, a reverse-phase HPLC
method was used to separate the lactic acid from the other labeled
products. Three radioactive peaks were observed, corresponding to
3-phosphoglyceric acid, glyceric acid, and lactic acid (Fig. 1). Glycerate presumably is formed by dephosphorylation
of a portion of the major product P-glycerate by traces of phosphatases
contaminating the enzyme preparations. The large enzyme concentrations
and extended assay periods necessitated by the feeble activity of
L8 amplifies the effects of even tiny traces of
phosphatases. No pyruvate (retention time 4.3 min) was apparent,
confirming the efficacy of the coupling system. The partitioning ratios
observed were 0.61 ± 0.05% (n = 6) and 0.71 ± 0.05% (n = 5) for L8 (Fig.
1A) and L8S8 (Fig. 1B),
respectively. The value for the wild-type enzyme is in agreement with
previous observations (16, 18).
Other Reaction Products of L8
Other side
reactions catalyzed by L8 and
L8S8 Synechococcus Rubiscos were
examined by observing the products derived from [1-14C]ribulose-P2. Anaerobic,
saturating-CO2 conditions were used. The labeled products
were separated by anion-exchange HPLC after borohydride reduction (16).
Partitioning between mono- and bis-phosphorylated sugar alcohols and
P-glycerate for the L8S8 enzyme was the same as
described previously (16) (Fig. 2A). For
L8, a fourth radioactive peak eluting just after
P-glycerate was also observed. Table I summarizes the
distribution of radioactivity between the various peaks for both enzyme
forms. The mono- and bisphosphate peaks from the L8
chromatogram were pooled and treated with alkaline phosphatase. The
sugar-alcohol moieties were then chromatographed on a
carbohydrate-separating column.
|
Three peaks of radioactivity were obtained for the sugar-alcohol moiety derived from the monophosphate fraction co-chromatographing with the D-ribulose 5-phosphate standard. The middle peak was broader and larger than the flanking peaks, consistent with it being composed of two overlapping peaks (Fig. 2B). None of the peaks coeluted with either ribitol, arabinitol, or xylitol, the expected products of reduction of pentuloses. The elution pattern was similar to that obtained for the reduced sugar moiety of the monophosphate by-product seen with the T65V mutant form of Synechococcus Rubisco. This by-product was identified as deoxypentodiulose-P (16). The unknown peak eluting near P-glycerate (Fig. 2A) co-chromatographs with a peak produced nonenzymatically from [1-14C]ribulose-P2 under strongly alkaline conditions (data not shown) that we have tentatively identified as being composed of both isomeric, branched, carboxylic acids produced by benzylic acid-type rearrangement of deoxypentodiulose-P (30). If this assignment is correct, then the radioactivity in this peak should be summed with that in the deoxypentodiulose-P peak. Since the combined total concentration of deoxypentodiulose-P and its putative rearrangement products at quenching was many fold greater than the concentration of enzyme protomers (Table I), these compounds must be genuine products, not merely the consequence of release of enzyme-bound enediol intermediate on quenching.
Reduction and dephosphorylation of the bisphosphate fraction remaining after exposure of [14C]ribulose-P2 to L8 yielded all three pentitols together with a small unknown peak frequently seen in controls lacking enzyme (Fig. 2C). Ribitol and arabinitol predominated, suggesting that the consumption of ribulose-P2 was incomplete as might be expected from the poor affinity of L8 for ribulose-P2 (19). Furthermore, the arabinitol/ribitol ratio was similar to that observed following reduction of ribulose-P2 in the minus-enzyme control, suggesting that little if any 3-ketoarabinitol-P2 had been produced (Table I). A small amount of xylitol was also observed, but its amount indicates that only a trivial amount of xylulose-P2 had been produced (Table I).
By-products of Catalysis by Rubisco Holoenzymes Under Conditions Leading to Exhaustion of Gaseous SubstratesUnder the usual CO2-saturating conditions, the Synechococcus holoenzyme produced negligible traces of by-products derived from the enediol intermediate, even when the reaction continued until the ribulose-P2 had been almost exhausted (Fig. 2A, Table I). In order to maximize the possibility of detecting enediol by-products with L8S8 holoenzymes, assay conditions were designed so that the gaseous substrates were exhausted well before complete consumption of ribulose-P2. The steady-state pool size of enzyme-bound enediol would be expected to be maximal under these conditions and, since carboxylating and oxygenating activities are suppressed by lack of substrates, the remaining ribulose-P2 would be exposed to large concentrations of enzyme for extended periods, amplifying the chance of detecting by-products. The data obtained (Table II) show that P-glycerate production ceased, as expected, when its concentration became approximately equal to the concentration of bicarbonate originally supplied. Given the high concentration of enzyme present, this would have occurred only seconds after initiation of the reactions. Subsequently, both spinach and Synechococcus holoenzymes converted the remaining ribulose-P2 to deoxypentodiulose-P and pentulose bisphosphate isomers of the substrate. Although production of these by-products was slow, readily measurable amounts had accumulated by the end of the experiments (Table II). While xylulose-P2 and 3-ketoarabinitol-P2 have been detected previously as trace products of spinach Rubisco (10-12), this is the first demonstration of the production of deoxypentodiulose-P by a wild-type Rubisco. In the case of the spinach enzyme, it is clear that deoxypentodiulose-P production represents true catalytic turnover because the amount of deoxypentodiulose-P produced far exceeded the concentration of active sites present. It is also possible that the deoxypentodiulose-P detected underestimates that actually produced because of further conversion of some of the deoxypentodiulose-P to other compounds, such as the rearrangement products mentioned above. These were not always detected as a discrete peak following the P-glycerate peak, as in Fig. 2A (solid line), and sometimes occurred as an unquantifiable tail on the P-glycerate peak (Fig. 2A, dashed line). For the Synechococcus holoenzyme, the deoxypentodiulose-P detected did not exceed the concentration of active sites present despite the longer incubation period.
|
Deoxypentodiulose-P formation during catalysis by spinach and
Synechococcus holoenzymes under conditions designed to lead to rapid exhaustion of gaseous substrate was also measured by observing
the absorbance at 330 nm of the quinoxaline adduct formed between
deoxypentodiulose-P and o-phenylenediamine (Fig.
3). For this experiment, it was important to exclude the
possibility that other -dicarbonyl species might also contribute to
the absorbance at this wavelength. Two other
-dicarbonyl species are
known to be produced by Rubisco side reactions. One is pyruvate which, like other
-keto acids (32), could form a quinoxaline adduct under
acidic conditions. However, this possibility was excluded by a control
experiment which showed that negligible increase in absorbance at 330 nm occurred when 0.5 mM pyruvate was added to this assay
system at pH 7.9 (Fig. 3). Another is the
-dicarbonyl by-product of
Rubisco's oxygenase reaction, detected with some mutants of R. rubrum Rubisco (23, 24). Use of strictly anaerobic conditions
avoided this second potential complication in the present experiments.
For comparison, P-glycerate production was measured in small aliquots
withdrawn at intervals from the cuvettes. For both enzymes, P-glycerate
production was initially rapid but ceased abruptly when the P-glycerate
concentration approached twice the concentration of NaHCO3
initially present, confirming
CO2/HCO3 exhaustion (Fig.
3). This indicates that o-phenylenediamine did not seriously
inhibit the carboxylase activity of either enzyme. The continuing rise
in absorbance at 330 nm confirmed that deoxypentodiulose-P was indeed
produced by both enzymes, supporting the conclusions from the analysis
of 14C-labeled products (Table II) and establishing beyond
doubt that this compound is a true by-product of both enzymes and not
solely the result of degradation of enediol intermediate released at quenching. Using the molar absorptivity estimated for the quinoxaline adduct (see "Experimental Procedures"), it may be calculated from the absorbance increase that approximately 1% of the added
ribulose-P2 had been converted to deoxypentodiulose-P by
the spinach enzyme in 20 min. Allowing for differences in enzyme
concentration and reaction time, this is in approximate agreement with
the amount of this by-product seen in the product analysis experiments
(Table II). No sign of a lag in the rate of absorbance increase was
observed with either enzyme during the early period before
CO2/HCO3
exhaustion.
CO2/HCO3
obviously caused
little suppression of deoxypentodiulose-P production, at least at the
moderately low concentrations initially present in these
experiments.
The rate of deoxypentodiulose-P production slowed progressively for both enzymes throughout the experiment (Fig. 3). For the spinach enzyme, this might be a result of inhibition caused by accumulating misprotonation by-products. Indeed, the kinetics of the decline for the spinach enzyme appeared quite reminiscent of the decline usually seen during catalysis which has been attributed to that cause (7-10). Synechococcus Rubisco, however, is not subject to progressive inactivation during catalysis, at least at CO2 saturation (16), and it showed a more pronounced decline such that, after 15-20 min, the rate of absorbance increase had fallen to approach the basal rate seen in the enzyme-free control. Therefore, accumulation of misprotonation products is not likely to be the cause of the inactivation in this case. Decarbamylation in the CO2-free conditions may be a more plausible reason.
The very slow increase in absorbance at 330 nm in the control lacking enzyme (Fig. 3) is consistent with known rate of spontaneous production of deoxypentodiulose-P from ribulose-P2 (30). Rapid conversion of the expected amount of ribulose-P2 to P-glycerate also engenders confidence that o-phenylenediamine does not quickly cause extensive ribulose-P2 degradation.
The discovery that mutagenic perturbation of
Rubisco's active site in the region that binds the P1 phosphate of the
substrate resulted in a tendency of the enediol intermediate to eliminate the P1 group (16, 17) raised a question about whether this tendency existed at all with wild-type Rubiscos and, if so, whether it
varied between different Rubiscos. Morell et al. (16)
speculated that the improvement in catalytic effectiveness under
limiting CO2 conditions that accompanied the evolution of
higher plant Rubiscos might have been achieved by increasing the
fraction of enzyme active sites in the enediol-bound form during
steady-state catalysis. If so, any tendency to
eliminate the
enediol intermediate would be more obvious with higher plant Rubisco
than with bacterial or cyanobacterial enzymes which are adapted to
higher CO2 concentrations. Experiments at saturating
CO2 with wild-type Rubisco showed little sign of the
elimination product, deoxypentodiulose-P (Table I). Therefore, to
address this question, it was necessary to devise experimental
conditions where the wild-type enzyme could be exposed to
ribulose-P2 for extended periods but prevented from disposing of it rapidly via carboxylation or oxygenation. This was
achieved under anaerobic conditions by the simple expedient of ensuring
that the starting concentration of ribulose-P2 was well in
excess of the CO2/HCO3
concentration. With sufficient Rubisco present to exhaust the inorganic
carbon supply (and any traces of O2) in seconds, residual ribulose-P2 would be left at a still saturating
concentration for the remainder of the experiment. With the main routes
of conversion of the enediol blocked by lack of gaseous substrates, the
active site would thus be expected to remain maximally charged with
enediol, maximizing its vulnerability to side reactions. This approach will only be successful if inactivation by decarbamylation does not
occur quickly in the absence of CO2. For the spinach enzyme at least, the data of Edmondson et al. (8) provide
confidence that decarbamylation is very slow under these conditions,
providing ribulose-P2 remains saturating. This confidence
is sustained by data which showed that deoxypentodiulose-P appeared in
reaction mixtures containing both spinach and Synechococcus
Rubiscos in easily detectable quantities under these conditions (Table
II, Fig. 3) but not in a control with decarbamylated enzyme (Table I,
footnote). Deoxypentodiulose-P formation was established both chromatographically, by resolving the products from
[1-14C]ribulose-P2 (Table II), and
spectrophotometrically, by measuring the formation of the quinoxaline
adduct with o-phenylenediamine (Fig. 3). Both methods agreed
that spinach Rubisco produced larger amounts of deoxypentodiulose-P
than Synechococcus Rubisco. Either spinach Rubisco has a
larger fraction of its active sites occupied by the enediol under these
conditions, as speculated by Morell et al. (16), or it
stabilizes or retains the enediol less effectively than
Synechococcus Rubisco. An idea of the scale of
deoxypentodiulose-P production can be obtained by comparing the amounts
observed with those of xylulose-P2, previously shown to be
produced by spinach Rubisco once in every 400 catalytic turnovers while
a constant supply of CO2 was maintained (10). In the
present experiments, spinach Rubisco produced approximately two-thirds
as much deoxypentodiulose-P as xylulose-P2, whereas
for the Synechococcus enzyme the ratio was approximately
one-fifth (Table II).
As expected from previous studies (10-12), spinach Rubisco also produced considerable amounts of pentulose bisphosphate isomers under these conditions. Our data show that the wild-type Synechococcus holoenzyme produces these isomers also, and in approximately similar amounts (Table II). The method for calculating the contributions of xylulose-P2 and 3-ketoarabinitol-P2 to the pool of substrate isomers assumes that these are the only two isomers produced. A third isomer, 3-keto-D-ribitol-1,5-bisphosphate, is also a theoretically possible misprotonation product although not shown so far to be the product of any Rubisco. If it is present, the method for calculating the amounts of individual pentulose bisphosphates collapses because it provides a potential second source of ribitol and xylitol. Nevertheless, the conclusion that pentulose bisphosphate isomers of the substrate, in general, are produced remains unassailed.
Absence of Small Subunits ExacerbatesThe
intrinsic tendency of the Synechococcus Rubisco holoenzyme
to catalyze elimination of its enediol intermediate is small enough
to be overlooked in experiments at saturating CO2 because of very rapid conversion of all of the ribulose-P2 supplied
to P-glycerate (Table I). In the absence of small subunits, however, the tendency becomes very large (Table I), rivalling that seen when
Thr-65 in the P1 binding site of the large subunit was mutated (16).
Thus it must be concluded that lack of small subunits also alters
active-site geometry in ways that impair Rubisco's ability to bind or
stabilize the enediol. This observation might provide some support for
the idea that binding of the small subunits indirectly influences the
arrangement of the P1-binding site within the active site (22).
Alternatively, since lack of small subunits is known to weaken the
affinity of the active site for ligands such as
2
-carboxyarabinitol-1,5-bisphosphate and to increase the
Km (ribulose-P2) (19), increased
elimination might simply reflect a weakening of the active site's grip
on the enediol with consequent decay of this intermediate after release into solution.
The impairment of the active site's ability to stabilize the enediol
intermediate induced by absence of small subunits was not accompanied
by a similar degradation of the ability to protect this intermediate
from misprotonation (Table I). Nor was the stability of the
aci-acid intermediate compromised by lack of the small
subunits because L8 partitioned its carboxylated
product between P-glycerate and pyruvate in a ratio similar to that of L8S8 (Fig. 1). Thus, although enolization and
processing of the six-carbon intermediate are impaired to approximately
equal extents by lack of small subunits (36), the impairment is quite
specific in its effect on side reactions; only elimination of the
enediol is selectively stimulated. Similar specificity is not apparent with mutant Rubiscos with enhanced enediol
elimination. Where examined, these mutants of both Synechococcus and R. rubrum Rubiscos also show either enhanced misprotonation of the
enediol and/or alterations in the amount of pyruvate produced (16, 17,
24, 37). The L8 data thus provide evidence that different
features within the active site must be involved in stabilizing the
enediol and aci-acid intermediates and that, even in the
case of the enediol intermediate, different features are involved in
suppressing its
elimination and its misprotonation.
We thank Nerida O'Shea for technical assistance and Gabriel Quinlan for preliminary measurements of pyruvate production by L8.