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J. Biol. Chem., Vol. 278, Issue 35, 32526-32536, August 29, 2003
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From the Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, P. O. Box 475, Canberra, Australian Capital Territory 2601, Australia
Received for publication, May 27, 2003 , and in revised form, June 3, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Leu-335 is located near the apex of loop 6 of the Rubisco large subunit,
which closes over the active site after ribulose-P2 binds
(6,
10). The adjacent residue,
Lys-334, is catalytically essential, and it makes contacts with the P1
phosphate and the C2 carboxylate formed by addition of CO2
(10), which stabilize the
transition state for carboxylation
(11). The Leu-335 residue
makes van der Waals contacts with the P2 phosphate (attached to C5 of the
substrate) (10). Thus the
effects of the Val-335 substitution were interpreted in terms of its
alteration of the position and orientation of the critical 
N of Lys-334
(8).
In addition to carboxylation and oxygenation, Rubisco also catalyzes other
reactions leading from the intermediates of the central two catalytic pathways
(Scheme 1)
(2). The initial step of
catalysis is the formation of an enediol (VI) from ribulose-P2 (V).
This enediol is subject to misprotonation to form xylulose-P2 (I)
and perhaps other pentulose bisphosphate isomers and 
elimination to
form deoxypentodiulose-P (II). The peroxyketone intermediate of the
oxygenation pathway (X) eliminates H2O2, producing
pentodiulose-P2 (XI), which rearranges to
carboxytetritol-P2 (XII). The aci-acid form of P-glycerate
(VIII), produced following C2/C3 cleavage of the carboxyketone (VII),
eliminates its phosphate group, forming pyruvate (III). Some of these side
reactions are enhanced by certain mutations
(12–17)
or when the small subunits are missing
(18). In one case, pyruvate
production was suppressed by a mutation
(12). By virtue of their
mimicry of ribulose-P2 or its enediolized form, the bisphosphate
by-products are slow binding, competitive inhibitors of Rubisco catalysis.
They have been implicated in the slow decline in Rubisco activity during
in vitro assay (the so-called "fallover" phenomenon) that
is always observed naturally with the higher plant enzyme
(19–26)
and can be induced mutationally in the Rhodospirillum rubrum enzyme
(27). Although
pentodiulose-P2 (XI) and carboxytetritol-P2 (XII) are so
far only known to be produced by mutant Rubiscos
(14,
15), the former is
particularly significant, because it is present in all ribulose-P2
preparations by virtue of spontaneous oxidation and will further accumulate by
this process during the usual aerobic Rubisco assays
(25). To mitigate fallover
inhibition of Rubisco in vivo, higher plants have Rubisco activase, a
motor protein powered by ATP hydrolysis that apparently manipulates the active
site of Rubisco to remove bound bisphosphates
(3,
28).
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Here we show that the perturbation of loop 6 caused by the Val-335 substitution affects catalysis more pervasively than can be explained by a simple misorientation of the adjacent Lys-334. The alteration exacerbates the inhibitor-producing side reactions, but the changed structure and dynamics of the loop permit the inhibitors to escape from, or be rearranged by, the active site. As a result, fallover inhibition during catalysis is suppressed or eliminated, depending on conditions, revealing the underlying causes of the phenomenon and suggesting how it may have evolved.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Measurement of Activity—Ribulose-P2 carboxylase
activity was determined at 25 °C using a modification of a
spectrophotometric assay (35).
Rubisco was activated at 50 °C for 10 min in 100 mM EPPS-NaOH
buffer solution, pH 8.0, containing 20 mM MgCl2, 1
mM EDTA, and 20 mM NaHCO3. Each assay
solution (2 ml) contained 100 mM EPPS-NaOH, pH 8.0, 20
mM MgCl2, 0.5 mM ribulose-P2, 0.5
mM dithiothreitol, 0.2 mM NADH, 2 mM ATP, 10
mM phosphocreatine, 20 mM NaHCO3, 0.1
mg·ml–1 carbonic anhydrase, 25
units·ml–1 creatine phosphokinase, 25
units·ml–1 3-phosphoglycerate kinase, 20
units·ml–1 glyceraldehyde phosphate
dehydrogenase, 55 units·ml–1 triose
phosphate isomerase, and 20 units·ml–1
glycerol phosphate dehydrogenase. The reaction was initiated by the addition
of activated Rubisco (3–12 nM active sites), and
A340 was measured at 5-s intervals for 2000 s. When
anaerobic conditions were required, the buffer was pre-sparged with
N2 before addition of 20 mM NaHCO3, and the
assays were carried out in septum-capped cuvettes with the headspace flushed
with N2. Characteristically of higher plant Rubisco
(19), activity declined
exponentially from an initial rate (
i) to reach a final,
steady-state rate (f). Data for product accumulation
versus time were fitted to the following equation
(19),
 | (Eq. 1) |
 | (Eq. 2) |
Xylulose-P2 carboxylase activity was measured using the same method, but with 25–50 µM xylulose-P2 instead of ribulose-P2, and using 0.5–2.0 µM active sites. Controls lacking Rubisco or ATP showed no activity.
Enolization of Ribulose-P2—The enolization rate was measured by NMR spectrometry by observing the rate at which the deuteron of [3-2H]ribulose-P2 was exchanged with a proton from solution. Assays were carried out at 25 °C in buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM EDTA, 10% (v/v) 2H2O, and 2.9 mM [3-2H]ribulose-P2. Reactions were initiated by the addition of Rubisco (to 3 µM sites) that had been activated as described above. Spectra were collected at 2-min intervals using a Varian INOVA 600-MHz NMR spectrometer. The water resonance was suppressed by the W5 WATERGATE pulse (36). The increasing intensity of the resonance at 4.37 ppm due to the 3-H proton with time was fitted to an exponential equation, and the initial rate was corrected for the 10% 2H2O present in the solvent. No exchange occurred in controls lacking enzyme.
Reaction Products—Assays were carried out at 25 °C in buffer containing 100 mM EPPS-NaOH, pH 8.0, 20 mM MgCl2, 1 mM EDTA, and 1 µM [1-3H]ribulose-P2. Assays under carboxylating conditions used buffer that had been sparged with N2 before the addition of 20 mM NaHCO3; assays under predominantly oxygenating conditions used buffer that had been sparged with O2 and had a final concentration of NaHCO3 (carried over with the preactivated enzyme solution) of 10 µM. Assays were initiated with preactivated enzyme (described earlier) to a final concentration of 3.7 µM active sites and terminated at 10 min after complete consumption of the ribulose-P2. To measure the total reaction products, the reaction was terminated by adding SDS to a final concentration of 2% (w/v). Denatured protein was removed using a Millipore Ultrafree-MC filter unit (Mr 10,000 cutoff) and the filtrate was loaded onto a Amersham Biosciences Mono Q 5/5 column equilibrated with 10 mM EPPS/10 mM boric acid/NaOH, pH 8.0, and the reaction products were separated using a NaCl gradient. The radioactively labeled compounds were detected by online scintillation counting (Ultima-Flo, Packard). The contributions of ribulose-P2 and xylulose-P2 to the unresolved pentulose bisphosphate peak were measured by collecting the peak, treating with alkaline phosphatase at pH 9.3, and carrying out chromatographic analysis of the pentuloses at 85 °C on a water-equilibrated Bio-Rad HPX-87C column. To measure products that remained bound to the enzyme, the reactions were terminated by ultrafiltration (as described above) before denaturation. The retentate was washed with four 400-µl aliquots of buffer before elution of the bound products with a 10 mM EPPS/10 mM boric acid/NaOH, pH 8.0, 2% (w/v) SDS and chromatographic analysis as described above. Radioactivity in the wash fractions was measured by scintillation counting.
Products that remained bound after complete consumption of oxidized ribulose-P2 were analyzed using a similar procedure. [1-3H]Ribulose-P2 (50 µM) was oxidized for 3 h with 1 mM CuSO4 as described (25). Oxidized [1-3H]ribulose-P2 (8 µM) was reacted at room temperature for 10 min with 10 µM preactivated Rubisco active sites in 50 mM EPPS-NaOH buffer, pH 8.0, 15 mM MgCl2, 1mM EDTA, 10 mM NaHCO3 before separation of Rubisco-bound radioactivity from unbound by gel filtration on a 1- x 20-cm column of Sephadex G-50 fine (Amersham Biosciences) equilibrated with the same buffer solution. The protein peak was collected, and the radioactivity it contained was measured and released by adding SDS to 2% (w/v). Denatured protein was removed by ultrafiltration, and an aliquot of the filtrate was chromatographed as described above. In some further experiments, 5 mM H2O2 was included in the solution during consumption of the oxidized ribulose-P2 by Rubisco; in others, addition of the H2O2 was delayed until 5 min after mixing the oxidized ribulose-P2 with Rubisco. Rubisco-bound radioactivity was separated by gel filtration and measured. The rate of pyruvate production during catalysis was measured spectrophotometrically as described (37) and compared with the rate of P-glycerate production measured with the standard assay described above.
Assays Under CO2 and O2
Limitation—Continuous spectrophotometric assays were conducted in
N2-sparged, septum-capped cuvettes with a minimal initial
concentration (50–100 µM), limited to that carried over
with the preactivated enzyme preparation (activated as described above), and
in the presence of excess ribulose-P2 (1 mM), so that
ample ribulose-P2 remained after
exhaustion. Deoxypentodiulose-P formation was measured from the absorbance of
its quinoxaline adduct with o-phenylenediamine as described by Morell
et al. (18) with a
final Rubisco concentration of 10 µM active sites used to
initiate the assay. Xylulose-P2 formation was measured using a
method based on that described by Edmondson et al.
(22) in a buffer solution
containing 100 mM EPPS-NaOH, pH 8.0, 20 mM
MgCl2, 1 mM EDTA, 1
mg·ml–1 rabbit muscle aldolase, 0.15
mg·ml–1 rabbit muscle glycerol phosphate
dehydrogenase, 0.2 mM NADH, and 1 mM
ribulose-P2. Reactions were initiated by addition of preactivated
Rubisco (final concentration, 4 µM active sites), and the rate
of decrease in A340 was measured. Both assays were conducted at 25
°C for 10 min after initiation, and rates of absorbance change in controls
lacking enzyme were subtracted.
Inhibition by Xylulose-P2—Rapid equilibrium inhibition by xylulose-P2 (competitive with respect to ribulose-P2) was measured as described (38) by adding preactivated enzyme (final concentration, 12 nM sites) to initiate spectrophotometric assays containing mixtures of ribulose-P2 (2.5–320 µM) and xylulose-P2 (0–40 µM). Initial activities for each substrate/inhibitor combination were measured, and the data for all combinations were fitted simultaneously to the hyperbolic equation for competitive inhibition to estimate the Km(ribulose-P2) and Ki(xylulose-P2).
Slow inhibition of initially fully carbamylated Rubisco was monitored using a method based on that of Zhu and Jensen (23). Rubisco (0.05–1 µM sites) was fully carbamylated by incubation with 100 mM EPPS-NaOH buffer, pH 8.0, 20 mM MgCl2, 20 mM NaHCO3, 1 mM EDTA. Then 0.4–500 µM xylulose-P2 was added, and, at intervals thereafter, assays were initiated by adding a 200-µl sample of this mixture to a 2-ml spectrophotometric assay mixture as described above. Initial activities were measured, the data were analyzed, and the various parameters were estimated as described below.
Inhibition by Carboxyarabinitol-P2—Binding of carboxyarabinitol-P2 to Rubisco was measured using the method of Pierce et al. (32), adapted for use with the spectrophotometric assay. Rubisco (10–50 nM sites) was fully carbamylated by incubation with 100 mM EPPS-NaOH buffer, pH 8.0, 20 mM MgCl2, 20 mM NaHCO3, 1 mM EDTA, and the other components required for the spectrophotometric assay (see above). Then 0.1–2.0 µM carboxyarabinitol-P2 was added and the mixture was incubated at 25 °C for various periods before assays were initiated by adding ribulose-P2 to 0.5 mM. The initial activities measured decayed exponentially as a function of the duration of incubation with carboxyarabinitol-P2. The data were analyzed, and the various parameters were estimated as described below. Release of carboxypentitol-P2 was measured by observing the exchange of enzyme-bound [14C]carboxypentitol-P2 with unbound, unlabeled carboxypentitol-P2 using a gel-filtration procedure to separate bound and unbound label (32).
Analysis of Slow Binding Inhibition—Both
xylulose-P2 and carboxyarabinitol-P2 are slow binding
inhibitors of Rubisco. This has been modeled previously as a two-step process
(32): an initial rapid
equilibrium interaction with the active site that competes with the substrate,
ribulose-P2, followed by a slow isomerization of the initial
complex to a much tighter complex that is likely to be associated with closure
of flexible loops of the protein over the ligand within the active site
(39).
 | (Eq. 3) |
 | (Eq. 4) |
 | (Eq. 5) |
is the Rubisco activity at time t, i is the
Rubisco activity at time zero, Ea (= [E] +
[EI]) and Et are the concentrations of
non-tightly complexed and total Rubisco active sites, respectively, I
is the inhibitor concentration, and Ki =
k2/k1. When k4 = 0
(i.e. the tight complex is non-exchangeable and inhibition proceeds
to completion, as it does with carboxyarabinitol-P2), these
relationships simplify to those used previously
(32); however, if
k4 is finite, inhibition proceeds until an equilibrium is
reached at which a portion of the enzyme remains unsequestered in the tight
complex and active. This active fraction is given by,
 | (Eq. 6) |
f is the final activity after preincubation to
equilibrium. Because of the saturation of the rate of inhibitor binding
inherent in the model (Equation
3), a residual active fraction persists even at saturating
concentrations of the slow binding inhibitor. This fraction is given by
k4/(k3 + k4).
Substituting Equation 6 into
Equation 4 leads to
Equation 2 and, by integration,
to Equation 1. These
relationships are analogous to those derived by Morrison
(40) for slow isomerization
binding. Rubisco was incubated for various periods with various concentrations of carboxyarabinitol-P2 or xylulose-P2, and its activity was measured immediately after mixing with ribulose-P2 as described above. To estimate Ki, k3, and k4, these data were fitted simultaneously to Equations 4 and 5, using multiple curve-fitting software (OriginLab, Northampton, MA).
Carbamylation of Lys-201 in the active site of Rubisco and subsequent binding of a divalent metal ion are prerequisites for catalysis (41). This introduces a complication into the model embodied in Equation 3, because some slow binding inhibitors, such as carboxyarabinitol-P2 (32), require the carbamylated, metal-complexed active site for tight binding, whereas others such as xylulose-P2 (23) bind more tightly to the uncarbamylated, metal-free site. This may cause k1 or k2, and thus Ki, to depend on the concentrations of CO2 and divalent metal, but, provided that these concentrations are kept constant and the binding and release of CO2 and divalent metal are rapid compared with inhibitor release, the above relationships remain applicable.
Activation of Initially Uncarbamylated, Metal-free Rubisco—The standard spectrophotometric assay (see above) was used to measure the rates at which uncarbamylated metal-free Rubisco became active after simultaneous exposure to Mg2+ ions, CO2, and ribulose-P2. The inactive complexes were formed by incubating 2.2–7.5 µM uncarbamylated Rubisco sites in 100 mM EPPS-NaOH buffer, pH 8.0, 1 mM EDTA with or without 80 µM xylulose-P2 for 30 min at 25 °C. To initiate activation, these solutions were added to complete assay mixtures containing 20 mM MgCl2, 20 mM NaHCO3, and 0.5 mM ribulose-P2 as described above (final Rubisco concentration, 150 nM sites). When the enzyme was preincubated with xylulose-P2 complex, the xylulose-P2 concentration carried over into the final assay solution was 400 nM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Enolization of Ribulose-P2 Is Only Slightly Impaired by the Val-335 Mutation—The Val-335 mutant Rubisco enolized [3-2H]ribulose-P2 at two-thirds of the rate of the wild-type enzyme (0.6 s–1, cf. 0.9 s–1, data not shown). This impairment is barely significant compared with the 6-fold reduction in the kcat value for carboxylation that the mutation causes (8).
The Val-335 Mutation Reduces Inactivation during
Catalysis—As is always observed with higher plant Rubiscos
(19,
24,
25,
43), the activity of the
wild-type Leu-335 Rubisco from tobacco declined progressively after addition
of ribulose-P2. However, this fallover was much less marked with
the Val-335 mutant enzyme (Fig.
1 and Table II).
Under aerobic conditions (Fig.
1A), the mutant enzyme lost activity very slowly
(kobs one-seventh of that of the wild type), losing only
14% of its initial activity in 500 s, compared with a 43% loss for the wild
type. Moreover, the decline of the mutant appeared to be qualitatively
different from that of the wild type. Consistent with previous reports
(19,
25), the data for the wild
type, when fitted to Equation 1, estimate a final steady-state rate (f) of 32% of the initial
rate (i). Similar fitting of the mutant's data project to a
f near zero, and this projection was confirmed in longer assays
(up to 8000 s, not shown).
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Under anaerobic conditions (Fig.
1B), shown previously to partially suppress fallover by
limiting oxidation of ribulose-P2
(25), the decline in activity
of the wild-type enzyme was considerably reduced in extent (f
was 74% of i) but occurred at a similar rate
(Table II). By contrast,
exclusion of O2 abolished fallover by the Val-335 mutant
completely.
Xylulose-P2 Carboxylase Activity Is Increased by the Val-335 Mutation—Consistent with previous observations (44, 45), wild-type tobacco Rubisco catalyzed the carboxylation of xylulose-P2 at a very slow rate (one six-thousandth of the rate with ribulose-P2, Fig. 1C and Table II). The kinetics of fallover with xylulose-P2 as substrate was quite similar to those with ribulose-P2. The Val-335 mutant catalyzed xylulose-P2 carboxylation faster than the wild type both in absolute terms (1.6-fold) and especially in terms of its ratio with respect to ribulose-P2 carboxylation (10-fold) (Table II). Furthermore, it did so with no signs of fallover. Indeed, an increase in xylulose-P2 carboxylase activity during xylulose-P2 carboxylation was observed consistently with the mutant enzyme (Fig. 1C).
The Val-335 Mutation Alters the Amounts of Side Reaction Products—The side-reaction product profile of the wild-type and mutant Rubiscos was observed by chromatographic analysis of the products of conversion of [1-3H]ribulose-P2 by a stoichiometric excess of Rubisco active sites under conditions, which promoted carboxylation or oxygenation (Figs. 2 and 3). In all cases, the conversion was virtually complete; only traces of ribulose-P2 (<0.5%) remained at the end of the reaction. Small amounts of some early-eluting non-phosphorylated compounds, which included pyruvate and products of trace phosphatase contamination, were poorly resolved by this procedure and were disregarded. Otherwise, all of the products of known side reactions were well resolved, except for the pentulose bisphosphates, which were resolved after dephosphorylation.
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Under carboxylating conditions, the Leu-335 wild-type enzyme produced labeled P-glycerate nearly exclusively, and none of it remained bound to the enzyme (Fig. 2, A and B). The Val-335 mutant enzyme produced mostly P-glycerate, along with a trace of carboxytetritol-P2 (0.3%), some of which remained bound (Fig. 2, C and D). When the assay conditions promoted oxygenase activity, Leu-335 produced mostly P-glycolate and P-glycerate (76 and 23%, respectively). Because the 3H label was attached to C1 of the ribulose-P2, the P-glycerate molecule produced by oxygenation was not labeled. Again, these products were fully released. Some labeled xylulose-P2 was also produced (0.3%), most of it remaining bound to the wild-type enzyme (Fig. 3, A and B). The resistance of this trace of bound xylulose-P2 to consumption by carboxylation implies that it must be bound to inactive, uncarbamylated enzyme likely to be abundant in these assays at low CO2 concentration. The increased oxygenation, relative to carboxylation, catalyzed by Val-335 Rubisco (8) was reflected in the ratio of [3H]P-glycolate (91%) to [3H]P-glycerate (7%) produced and released. The mutant enzyme also produced several side products: pentodiulose-P2 (0.4%), xylulose-P2 (0.4%), and carboxytetritol-P2 (0.8%), the latter remaining substantially bound to the enzyme (Fig. 3, C and D). With the mutant, the xylulose-P2 produced did not remain bound.
Other experiments (not shown) revealed that pentodiulose-P2 was
not fully retained on the enzyme during the long period (
2 h) required
for the centrifugal ultrafiltration and washing procedure used to separate
bound products in these experiments (Figs.
2 and
3). This caused variable
retention of labeled pentodiulose-P2. Either this species is slowly
released itself (consistent with the reported Kd of 0.13
µM (25)) or it
might be slowly converted to monophosphate products on the active site before
release. Therefore, the amounts of bound pentodiulose-P2 detected
in those experiments must be considered to be minimum estimates.
Carboxytetritol-P2 appeared to be better retained.
The rate of production of pyruvate, a by-product of the carboxylation
reaction resulting from -elimination of the aci-carbanion
intermediate (Scheme 1) was
measured spectrophotometrically under carboxylating conditions. In agreement
with previous observations with other Rubiscos
(37), the Leu-335 wild-type
produced pyruvate at 0.69% of the rate at which it produced pairs of
P-glycerate molecules. However, the analogous ratio for the Val-335 mutant was
only one-fifth as great (0.13%).
Binding and Processing of Ribulose-P2 Oxidation
Products—As reported previously
(25), two of the side-reaction
products seen with the Leu-335 mutant enzyme, pentodiulose-P2 and
its product of benzylic acid-type rearrangement, carboxytetritol-P2
(Scheme 1), can be produced
non-enzymatically by oxidation of ribulose-P2 in the presence of
Cu2+ ions. Chromatographic resolution of the oxidation
products (Fig. 4A)
revealed the presence of both of these compounds, together with a variety of
monophosphorylated and non-phosphorylated compounds arising from further
degradation of the unstable pentodiulose-P2
(25). This mixture of
oxidation/degradation products, including 40% residual unoxidized
ribulose-P2, was reacted with Leu-335 and Val-335 Rubiscos to
completion under carboxylating conditions. Unbound compounds were then removed
by gel filtration, as quickly as possible (within 20 min) to maximize
retention of bound pentodiulose-P2. The unbound compounds were
almost entirely monophosphate species (data not shown). With both Rubiscos,
the fraction of radioactivity that remained bound correlated approximately
with the total pentodiulose-P2 plus
carboxytetritol-P2 in the oxidized mixture
(Table III). Further
information about the identity of the bound species was sought by adding
H2O2, which is known to cleave Rubisco-bound
pentodiulose-P2 and release the cleaved products, P-glycolate and
P-glycerate (25). With both
wild-type and mutant enzymes, adding H2O2 simultaneously
with the oxidized ribulose-P2 preparation eliminated over 75% of
the bound radioactivity. When the H2O2 was added to the
reaction mixture 5 min after the oxidized ribulose-P2 preparation,
when conversion to products had been completed, the result was similar with
the wild-type Rubisco but different with the Val-335 mutant, which retained
80% of the radioactivity bound in the absence of H2O2
(Table III). Apparently, at
least two compounds derived from oxidized ribulose-P2 must remain
bound to Rubisco in these experiments. One is released from the enzyme by
H2O2, and the other is not, and the former is
substantially converted to the latter by the mutant enzyme, but not by the
wild type. This conclusion was confirmed and the two compounds were identified
as pentodiulose-P2 and carboxytetritol-P2 by
anion-exchange chromatography of the radioactivity bound in the absence of
H2O2 and released by treatment with SDS. Whereas the
wild-type enzyme bound and retained pentodiulose-P2 and
carboxytetritol-P2 approximately in the same proportions as they
existed in the oxidized ribulose-P2 preparation
(Fig. 4B), the
proportions were reversed with the mutant, which had catalyzed the benzylic
acid-type rearrangement of most of the pentodiulose-P2 present in
the oxidized ribulose-P2 to carboxytetritol-P2
(Fig. 4C). Obviously,
the mutation must alter the structure of the active site in a way that
facilitates this rearrangement.
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The Val-335 Mutation Promotes Side Reactions Originating from the
Enediol Intermediate—Although the rates of "misfire"
reactions originating from the enediol intermediate
(Scheme 1 (12,
13,
18,
22,
23)) are small compared with
the rate of CO2 addition to this intermediate, they can be measured
in the absence of CO2 and O2 where consumption of
ribulose-P2 by carboxylation and oxygenation is prevented and the
fraction of the enzyme with enediol bound is maximized. Although
CO2 is required to activate Rubisco by carbamylation, this
requirement can be fulfilled by preincubation in a
-containing solution. When a small
aliquot of preactivated enzyme is subsequently added to the assay mixture, the
final is small and rapidly
exhausted by carboxylation. Decarbamylation of higher plant Rubisco in
CO2-free medium occurs very slowly while ribulose-P2
persists (20). This device
readily allows spectrophotometric measurement (see "Experimental
Procedures") of both elimination of the enediol to produce
deoxypentodiulose-P and misprotonation to produce xylulose-P2. Both
assays showed an initial lag (presumably caused by the coupling systems and
consumption of the last traces of
)
followed by a near linear period. When expressed as percentages of the
carboxylation kcat, both activities were substantially
increased by the Leu-335 mutation (10-fold for deoxypentodiulose-P and 4-fold
for xylulose-P2, Table
IV).
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Inhibition by Xylulose-P2—Fully CO2/Mg2+-activated wild-type Rubisco was slowly inhibited by exposure to xylulose-P2 (Fig. 5A). Similar slow inhibition was seen with spinach Rubisco by Zhu and Jensen (23) who attributed it to the formation of a decarbamylated enzyme-xylulose-P2 complex. We analyzed xylulose-P2 inhibition in terms of the model for slow binding inhibition (Equation 3) (40) used previously to model inhibition of Rubisco by carboxyarabinitol-P2 (32, 42). The rate constants for slow binding and release, k3 and k4, were obtained by fitting data for the rate of inhibition at varying xylulose-P2 concentrations to Equations 4 and 5. The results obtained show that, although the Ki values for the rapid-equilibrium steps are similar for xylulose-P2 and carboxyarabinitol-P2 binding, there are striking differences in the rate constants for the subsequent isomerization step (Table I). First, the isomerization step for xylulose-P2 binding is very slow (approximately one-hundredth of the rate of the analogous step for carboxyarabinitol-P2 binding); second, xylulose-P2 is released from its tight complex at least 5000-fold faster than is carboxyarabinitol-P2; and third, although the Val-335 mutation perturbs the kinetics of carboxyarabinitol-P2 only modestly, it completely abolishes the slow isomerization phase of xylulose-P2 binding so that no slow inhibition was seen, even after prolonged incubation at xylulose-P2 concentrations 20-fold greater than its rapid equilibrium Ki (Fig. 5).
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As predicted for slow isomerization binding where release from the tight
complex occurs at an appreciable rate relative to isomerization
(Equation 3), inhibition of the
wild-type, Leu-335 Rubisco by xylulose-P2 plateaued at less than
complete inhibition. A fraction of activity was retained (25% in this case),
even after prolonged incubation to equilibrium at saturating
xylulose-P2 concentrations (Fig.
5B). Although the data for
f/i versus xylulose-P2
concentration may be fitted to Equations
5 and
6, the value of the parameter
estimates obtained by this method is limited by strong interdependency between
k3 and k4. Nevertheless, a curve drawn
using the estimates of these parameters obtained from the data of
Fig. 5A fits these
data approximately (Fig.
5B).
The data reported in Fig. 5
are the initial activities seen immediately after 10-fold dilution of
xylulose-P2-treated ECM preparations into the assay solution
containing saturating ribulose-P2. Under these assay conditions,
reactivation slowly occurred. In all cases, this recovery proceeded with a
kobs of 7 x 10–4
s–1 (data not shown), which is similar to the
k4 value estimated from the rate of xylulose-P2
inhibition (Table I), as
expected.
The action of xylulose-P2 as a rapid equilibrium inhibitor, competitive with respect to ribulose-P2, was assessed by measuring the activity of preactivated Rubisco immediately after addition to assay mixtures containing various ribulose-P2 and xylulose-P2 concentrations. Both wild-type and mutant enzymes showed classic, competitive inhibition. The Ki for xylulose-P2 was 4.8 µM for the Leu-335 form, which is consistent with the value reported for spinach Rubisco (38), and agrees approximately with the estimate obtained from the slow binding experiment (Fig. 5A). For the Val-335 form, the estimate increased to 20.7 µM (Table I).
Activation of Uncarbamylated Rubisco in the Presence of Xylulose-P2—When uncarbamylated, metal-free Rubisco (E) is added to an assay mixture containing saturating concentrations of CO2, Mg2+, and ribulose-P2, activity accelerates as the essential Mg2+-stabilized carbamate (ECM) forms in the active site. Although the rate of ECM formation is slowed by the presence of ribulose-P2, which binds tightly to E (46), activity nevertheless increases from an initial zero to a final steady state where it matches that seen in controls where full CO2/Mg2+ activation (ECM formation) was induced before addition of ribulose-P2. The observed rate constant for this activation was increased slightly by the Val-335 mutation (Fig. 6), perhaps indicating that ribulose-P2 binds a little less tightly to the E form of the mutant than the wild type.
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The tendency of ligands, such as xylulose-P2, to bind to the uncarbamylated E form of Rubisco may be gauged by observing the effect of preincubation of E with the ligand on the rate of activation in the above assay. The substantially reduced rate of activation caused by pre-exposure of the Leu-335 wild type to xylulose-P2 (Fig. 6A) indicates that xylulose-P2 binds to E and is released from it much more slowly than the rate of carbamylation under the same conditions without preincubation with xylulose-P2. Similarly slow release from the E-xylulose-P2 complex was reported for spinach Rubisco (23, 44). The kobs for xylulose-P2 release from the wild-type tobacco enzyme estimated by this means (Fig. 6A) is similar to the release parameter, k4, estimated from the rate of xylulose-P2 inhibition of the ECM form (Table I). However, in the experiment shown in Fig. 6A, we can be sure that the starting EI* species is uncarbamylated. Therefore, this observation lends support to the view that the tight complex with xylulose-P2 is uncarbamylated, regardless of the carbamylation status of the starting, uninhibited enzyme (23, 44). In marked contrast, the effect of preexposure to xylulose-P2 on subsequent activation of the Val-335 mutant was barely perceptible (Fig. 6B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Val Substitution Does Not Grossly Disturb
the Active Site of Rubisco—Consistent with previous assessments
(8), the Val-335 mutant of
tobacco Rubisco is properly assembled and fully soluble. It crystallizes
easily and can be purified in quantity by the same procedures used for the
wild-type protein. Its apparently unimpaired tight binding of the reaction
intermediate analog, carboxyarabinitol-P2, and its only slightly
impaired capacity for enolization of the substrate indicate that perturbation
of the active by the mutation must be quite subtle. Slow Inhibition during Catalysis Is Less Severe and Different in Character with Val-335 Rubisco—Consistent with the view that fallover inhibition of Rubisco during catalysis is caused by accumulation of slow binding inhibitors at the active site (21, 22), assay time-course data (Fig. 1) fit well to Equation 1, which can be derived from the two-step, slow isomerization model (40) of slow inhibition (see "Experimental Procedures"). However, interpretation of the meanings of the kinetic parameters estimated in this way is clouded by observations that at least two different inhibitors are responsible and their concentrations vary during the assay because they are largely generated during the course of the assay itself, either by Rubisco-catalyzed misprotonation of the enediolate intermediate (22) (Scheme 1) or by non-enzymatic oxidation of ribulose-P2 (25).
In the presence of atmospheric concentrations of O2, the Val-335
mutation greatly reduced the severity of fallover inhibition and altered its
character in the sense that the inhibition proceeded to completion
(i.e. f 0) rather than coming to a steady state
where a finite fraction of the activity remained, as with the wild type
(Fig. 1A). This
suggests that the residual inhibition seen with the Val-335 mutant is caused
by an inhibitor whose rate of release (k4,
Equation 3) from the tight
complex (EI*) is close to zero, unlike wild-type fallover where the
finite steady-state f indicates that k4
must be appreciable compared with k3.
Exclusion of O2 reduced the extent of fallover inhibition of the wild-type enzyme (Fig. 1B), consistent with previous observations that non-enzymatic oxidation of ribulose-P2 to pentodiulose-P2 occurring during the assay was one of the main causes of the fallover phenomenon (25). Total abolition of fallover inhibition of the Val-335 mutant enzyme under the same conditions (Fig. 1B) is consistent with pentodiulose-P2, or a product derived from it, being the sole cause of the residual inhibition seen with the mutant under aerobic conditions.
All Abortive Side Reactions, Except Pyruvate Production, Are
Exacerbated by the Val-335 Mutation—Obviously the reduced fallover
inhibition seen with the mutant is not a result of reduced production of
inhibitory by-products; production of all of these was increased by the
mutation, except pyruvate. Another active site mutation, Thr-65 Val in
Synechococcus PCC6301 Rubisco, also partitioned less product toward
pyruvate (12). Apparently,
loosening the active site or reducing the catalytic flux can, in some
circumstances, aid the stereospecific protonation that completes the
carboxylation sequence (Scheme
1).
The increased tendency to catalytic misfire induced by the mutation was
measured most quantitatively with the by-products arising from the enediolate
intermediate, xylulose-P2 and deoxypentodiulose-P, because
spectrophotometric assays can be devised
(Table IV). The 4- and 10-fold
increases in the partitioning of product toward these compounds attest to the
impairment of the ability of the mutant enzyme to control proton access to the
Re face of C3 of the enediolate and to maintain the planarity of the
O1, C1, C2, and C3 atoms that is so essential for suppressing
elimination of the P1 phosphate
(2). The monophosphate
deoxypentodiulose-P is not likely to be a strong inhibitor of Rubisco, but the
decreased fallover inhibition, despite enhanced partitioning toward
xylulose-P2, suggests that the interaction of this bisphosphate
with the active site must be considerably loosened by the mutation.
Further information about misfire products was obtained by chromatographic analysis of total and enzyme-bound products after complete conversion of labeled ribulose-P2 (Figs. 2 and 3). Here the gradient of the anion-exchange chromatography was optimized to focus attention on the bisphosphate and carboxylated-monophosphate products. The experiment under predominantly oxygenating conditions (Fig. 3) was the most informative, because the low CO2 concentration maximized the fraction of active sites with enediol bound, and thus the side reactions that stem from this intermediate. Furthermore, the high O2/CO2 ratio promotes flux through the oxygenation pathway, allowing detection of the pentodiulose-P2 and carboxytetritol-P2 products that derive from the peroxyketone intermediate (Scheme 1). Xylulose-P2 was the only bisphosphate by-product arising from the Leu-335 wild type (Fig. 3A), much of it remaining bound to the enzyme (Fig. 3B). The Val-335 mutant also produced pentodiulose-P2 and carboxytetritol-P2 (Fig. 3C) with only the latter remaining bound (Fig. 3D). This supports the view that xylulose-P2 is not bound tightly by the mutant enzyme. It also demonstrates that the mutant does not fully suppress H2O2 elimination from the peroxyketone intermediate but is able to promote the benzylic acid-type rearrangement of the elimination product, pentodiulose-P2, to carboxytetritol-P2 (Scheme 1), which remains tightly bound at the active site. Enhanced production of pentodiulose-P2 and carboxytetritol-P2 was observed previously with mutants of R. rubrum Rubisco (14, 15).
The Val-335 Mutation Promotes Rearrangement of
Pentodiulose-P2 to Carboxytetritol-P2 within the Active
Site—Exposure to Cu-oxidized ribulose-P2, which contains
pentodiulose-P2 and carboxytetritol-P2
(25), provided a more direct
demonstration of the ability of the active site to bind
pentodiulose-P2 and, in the case of the mutant, to rearrange it to
carboxytetritol-P2. These experiments benefited from the
recognition that, unlike carboxytetritol-P2,
pentodiulose-P2 is not stable when bound to Rubisco and, therefore,
rapid isolation of the complex by gel filtration is required for optimal
detection. These experiments also exploited the ability of added
H2O2 to return pentodiulose-P2 to the
catalytic pathway for oxygenation, leading to its release from the enzyme as
P-glycolate and P-glycerate
(25). The results
(Table III and
Fig. 4) demonstrate clearly
that both wild-type and mutant enzymes bound both pentodiulose-P2
and carboxytetritol-P2 and that the mutant converted the former to
the latter with much greater facility than the wild type. Similar facilitation
of this benzylic acid-type rearrangement was observed previously with the
Lys-329 Ala (analogous to residue 334 in the higher plant enzyme)
mutant of R. rubrum Rubisco
(15).
Slow Binding Inhibition of Rubisco—The data (Figs. 5 and 6 and Table I) are consistent with xylulose-P2 being a slow binding inhibitor following the classic two-step, slow isomerization model (Equation 3) (40). Its inhibition is analogous to the more thoroughly studied inhibition by 2'-carboxyarabinitol-P2 and 4'-carboxyarabinitol-P2 (32, 42) but differs in that it is two orders of magnitude slower and that it has an appreciable release rate, so that it comes to an equilibrium where 25% of the activity remains uninhibited even at saturating xylulose-P2 concentrations. Whether the slow rate of inhibition is related to the preference of xylulose-P2 for binding to the uncarbamylated E form of the active site (23, 44) remains to be determined. This could be assessed by comparison with another slow binding inhibitor, such as 2'-carboxyarabinitol-1-phosphate, which is known to prefer the carbamylated site (ECM) (47) but binds less tightly than carboxyarabinitol-P2 (48). Measurement of the rate of slow inhibition by xylulose-P2 commencing with E rather than ECM (as in Fig. 5) also might shed light on this question.
Saturation of inhibition by xylulose-P2 at less than complete inhibition was observed previously (23, 38), and this phenomenon is shared by one or more inhibitors remaining in Rubisco assays after complete consumption of ribulose-P2 (21), 2'-carboxyarabinitol-1-phosphate (48, 49), and pentodiulose-P2 (25). Although this might suggest anti-cooperativity (i.e. binding of the ligand to one or more sites on a Rubisco octamer makes binding to subsequent sites less favored) (21), it is more economically explained as being simply the expected consequence of the model for slow inhibition embodied in Equation 3, where a rate of inhibition that becomes saturated at high inhibitor concentrations is coupled with an appreciable rate of release (see "Experimental Procedures").
Alterations to Fallover Inhibition Caused by the Val-335 Mutation—Two different slow binding inhibitors have been definitely implicated in fallover inhibition: xylulose-P2 produced by misprotonation of the enediolate (22) and pentodiulose-P2 produced by non-enzymatic oxidation of ribulose-P2 during its synthesis and storage and also under the conditions of the Rubisco assay (25). Another pentulose bisphosphate misprotonation product, 3-keto-D-arabinitol-1,5-bisphosphate, has been hypothesized to contribute to fallover inhibition (18, 22, 24, 50). However, its identification as its arabinitol product after dephosphorylation and borohydride reduction is compromised by observations that pentodiulose-P2 can generate predominantly the same product after similar treatment (14), presumably because of stereochemical bias during reduction. Therefore, we do not consider that the contribution of 3-keto-D-arabinitol-1,5-bisphosphate to fallover inhibition has been established securely. The observation, that production of putative 3-keto-D-arabinitol-1,5-bisphosphate depended on the presence of O2 (50), heightens suspicion that the species detected was really the ribulose-P2 oxidation product, pentodiulose-P2. If so, xylulose-P2 and pentodiulose-P2 may be the only significant causes of fallover inhibition.
One of the central observations of this study is the total abolition of the slow isomerization phase of xylulose-P2 inhibition by the Val-335 mutation. Rapid equilibrium inhibition by this species still persists in weakened form (Table I), but no sign of the second step of the two-step binding mechanism (Equation 3) was evident. Apparently, the minimal perturbation of loop 6 in the mutant (effectively, removal of one methylene carbon) was sufficient to loosen its interactions with the active site and allow the rapid release of the ligand. The binding of other slow binding bisphosphates, such as pentodiulose-P2, might be similarly affected, but the instability of the latter compound precludes detailed study.
When slow inhibition by xylulose-P2 (and perhaps 3-keto-D-arabinitol-1,5-bisphosphate, if it exists) was prevented by the Val-335 mutation and pentodiulose-P2 production was blocked by anoxia, fallover was abolished completely (Fig. 1B), a situation never encountered previously with any higher plant Rubisco. Four pieces of circumstantial evidence collectively mount a strong case that carboxytetritol-P2 is the inhibitor responsible for the reduced degree of fallover inhibition retained by the mutant under aerobic conditions (Fig. 1A). First, unlike the wild-type enzyme, the mutant catalyzes the production of significant amounts of pentodiulose-P2, the precursor of carboxytetritol-P2, when O2 is present (Figs. 2 and 3). This substantially augments the basal amounts of this dicarbonyl compound produced by non-enzymatic oxidation during the course of the assay, the lack of which causes the moderate alleviation of fallover inhibition of the wild-type enzyme in anoxia (Fig. 1, A and B) (25). Second, the mutation greatly facilitates the benzylic acid-type rearrangement of pentodiulose-P2 to carboxytetritol-P2 (Fig. 4 and Table II). Third, bound carboxytetritol-P2 must be released from the active site imperceptibly slowly, despite the loosening of loop 6 induced by the mutation. This is shown by the recovery of enzyme-bound carboxytetritol-P2 even after four-times repeated washing by centrifugal ultrafiltration over a 2-h period, a procedure that led to a loss of most of the bound pentodiulose-P2 (Figs. 2 and 3). Fourth, consistent with this, the residual fallover inhibition displayed by the mutant projects to complete inhibition, suggesting that it is caused by an inhibitor whose release rate (k4, Equation 3) is close to zero.
The Val-335 mutation therefore illustrates the relative contributions of the two main causes of fallover inhibition by eliminating one (by facilitating rapid release of any pentulose bisphosphates produced within the active site by misprotonation) and exacerbating the other when O2 is present (by permitting rearrangement of pentodiulose-P2 to carboxytetritol-P2, which binds even more tightly). Reduced fallover inhibition of spinach Rubisco observed after chemical modification of some Arg residues with phenylglyoxal (26) might reflect similar facilitation of pentulose bisphosphate release from active sites indirectly perturbed by modification of remote Arg residues.
Alterations to Xylulose-P2 Carboxylation—The increased capacity to catalyze xylulose-P2 carboxylation induced by the mutation (Fig. 1C and Table II) presumably is the consequence of looser packing of loop 6 against the substrate. By some means, this apparently allows an increase in the rate of abstraction of the C3 proton from xylulose-P2, which, if xylulose-P2 binds exactly analogously to ribulose-P2, would not be positioned appropriately for abstraction by carbamyla
, Val-335, every tenth 5-s data
point shown) versus time were fitted to
,
, 
, and 
) and Val-335 (