Plant Physiol. (1998) 117: 1059-1069
Potent Inhibition of Ribulose-Bisphosphate Carboxylase by an
Oxidized Impurity in Ribulose-1,5-Bisphosphate1
Heather J. Kane,
Jean-Marc Wilkin2,
Archie R. Portis Jr., and
T. John Andrews*
Research School of Biological Sciences, Australian National
University, P.O. Box 475, Canberra ACT 2601, Australia (H.J.K.,
J.-M.W., T.J.A.); and Photosynthesis Research Unit, Agricultural
Research Service, United States Department of Agriculture, Urbana,
Illinois 61801 (A.R.P.)
 |
ABSTRACT |
Oxidation
of D-ribulose-1,5-bisphosphate (ribulose-P2)
during synthesis and/or storage produces
D-glycero-2,3-pentodiulose-1,5-bisphosphate (pentodiulose-P2), a potent slow, tight-binding inhibitor
of spinach (Spinacia oleracea L.)
ribulose-P2 carboxylase/oxygenase (Rubisco). Differing
degrees of contamination with pentodiulose-P2 caused the
decline in Rubisco activity seen during Rubisco assay time courses to
vary between different preparations of ribulose-P2. With
some ribulose-P2 preparations, this compound can be the
dominant cause of the decline, far exceeding the significance of the
catalytic by-product, D-xylulose-1,5-bisphosphate. Unlike
xylulose-1,5-bisphosphate, pentodiulose-P2 did not appear
to be a significant by-product of catalysis by wild-type
Rubisco at saturating CO2 concentration. It was produced
slowly during frozen storage of ribulose-P2, even at low
pH, more rapidly in Rubisco assay buffers at room temperature, and
particularly rapidly on deliberate oxidation of ribulose-P2 with Cu2+. Its formation was prevented by the exclusion of
transition metals and O2. Pentodiulose-P2 was
unstable and decayed to a variety of other less-inhibitory compounds,
particularly in the presence of some buffers. However, it formed a
tight, stable complex with carbamylated spinach Rubisco, which could be
isolated by gel filtration, presumably because its structure mimics
that of the enediol intermediate of Rubisco catalysis. Rubisco
catalyzes the cleavage of pentodiulose-P2 by
H2O2, producing P-glycolate.
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INTRODUCTION |
The photosynthetic CO2-fixing enzyme Rubisco
catalyzes both carboxylation and oxygenation of
ribulose-P2, producing either two molecules of
P-glycerate or a molecule each of P-glycerate and P-glycolate. The
catalytic reaction proceeds via enolization of
ribulose-P2 following abstraction of the proton
attached to C-3 by an enzymatic base. This is followed by the
attack of CO2 or O2 on C-2
of the resultant 2,3-enediol. Depending on whether the attacking
species is CO2 or O2,
either 2
-carboxy-3-keto-arabinitol-1,5-bisphosphate or
2-peroxy-3-keto-arabinitol-1,5-bisphosphate is formed as
enzyme-bound intermediates. These intermediates are hydrated and
cleaved between C-2 and C-3 to yield the products (for review,
see Andrews and Lorimer, 1987
; Hartman and Harpel, 1994; Gutteridge and
Gatenby, 1995; Cleland et al., 1998).
The activity of higher-plant Rubisco declines during assay in vitro.
The decrease in activity is approximately exponential and commences as
soon as fully activated Rubisco is mixed with ribulose-P2. Depending on the conditions, it
proceeds with a half-time of 5 to 10 min and eventually reaches an
apparent steady state in which the final activity is 20 to 50% of the
initial activity. This phenomenon, now generally called "fallover,"
has been explained in terms of the production of isomers of
ribulose-P2, xylulose-P2, and ketoarabinitol-P2 by stereochemically
incorrect reprotonation of the enediol intermediate (Edmondson et al.,
1990a, 1990b, 1990c, 1990d; Zhu and Jensen, 1991a, 1991b). However, two
observations suggest that isomer production can explain fallover only
partially. First, millimolar concentrations of
H2O2 alleviate the decline in activity. Although H2O2
inhibits the initial rate, the subsequent decline occurs more slowly
(Badger et al., 1980; Edmondson et al., 1990a). Since such
concentrations of H2O2 do
not rapidly destroy pentulose bisphosphates, this observation is
difficult to explain in terms of inhibition by these isomers. Second,
the rate and extent of the activity decline can vary between different preparations of ribulose-P2, and this is
inconsistent with inhibition by reaction by-products. Both of these
observations might be consistent with the variable presence of an
inhibitor of Rubisco in ribulose-P2 preparations
if that inhibitor was a slow, tight binder and if it was destroyed by
H2O2.
A clue about the possible identity of such an
inhibitor was provided by observations with certain site-directed
mutants of Rhodospirillum rubrum Rubisco that were severely
disabled in their ability to promote carboxylation of the enediol
intermediate (Chen and Hartman, 1995; Harpel et al., 1995b). These
mutants produced by-products of the oxygenase reaction that were
identified as pentodiulose-P2 and its product of
benzylic-acid-type rearrangement, carboxytetritol-P2.
Pentodiulose-P2 has a vicinal dicarbonyl function that renders it sensitive to cleavage by
H2O2, and it is possible that it might also be formed from ribulose-P2 by
nonenzymatic oxidation.
This study was motivated by frustration with the variable quality of
ribulose-P2 preparations. Both of our
laboratories have experienced ribulose-P2
preparations that for no obvious reason have induced such rapid and
extensive declines in activity of higher-plant Rubisco that the
preparations were unusable. We undertook this study to investigate the
identity and source of potential Rubisco inhibitors in the hope of
discovering means of suppressing their formation.
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MATERIALS AND METHODS |
Materials
[1-14C]Ribulose-P2
was synthesized and purified according to the method of Kane et al.
(1994) and stored at pH 2.8 and at 
80°C for approximately 4 years
before use. This preparation contained impurities arising from decay of
ribulose-P2 during storage (see ``Results'').
[1-3H]Ribulose-P2 was
synthesized from [2-3H]Glc (Amersham) using the
same method but purified by anion-exchange chromatography on a Mono-Q
5/5 column (Pharmacia) equilibrated with 3 mM HCl, using a
shallow, linear NaCl gradient rising at 20 mM column
volume
1 from 0 to 300 mM. To remove
NaCl, pooled fractions were concentrated 10-fold in vacuo and then gel
filtered at 4°C on Sephadex G-10 equilibrated with 3 mM
HCl. Ribulose-P2-containing fractions were pooled, snap frozen, and stored in liquid N2.
This preparation was free of impurities initially and showed no signs
of them even after storage for 1 year in liquid
N2. Unlabeled ribulose-P2
was synthesized from Rib-5-phosphate (Sigma) according to the method of
Horecker et al. (1958) and purified on a 4.4- × 100-cm column of
AG1-X8 (Cl form, Bio-Rad) equilibrated with 3 mM HCl, using a linear NaCl gradient rising at 24 mM column volume1 from 100 to 250 mM. Pooled fractions containing
ribulose-P2 that eluted at 150 mM
NaCl were snap frozen in small aliquots and stored in liquid
N2.
Rubisco was purified from spinach (Spinacia oleracea
L.) (Edmondson et al., 1990a; Morell et al., 1997) and
Rhodospirillum rubrum (Andrews and Kane, 1991) as
described previously. Before use, spinach Rubisco was dialyzed
at 4°C overnight against 50 mM Hepps
(N-2-hydroxyethylpiperazine-N-3-propanesulfonic
acid)-NaOH buffer, pH 8.0, containing 15 mM
MgCl2, 1 mM EDTA, and 10 mM NaHCO3, and then incubated at
50°C for 10 min and rapidly cooled.
NaH14CO3 was supplied by
Amersham, and scintillant, either Emulsifier Safe or Ultima Gold XR, by
Canberra Packard. Sigma supplied catalase (bovine liver), creatine
kinase (rabbit muscle), and carbonic anhydrase (bovine erythrocyte).
All other enzyme preparations were obtained from Boehringer Mannheim as
saturated
(NH4)2SO4 suspensions and desalted before use.
Rubisco Fallover Assays
Rubisco assays were conducted aerobically (unless otherwise
stated) for extended periods at 25°C in assay solutions containing 100 mM Hepps-NaOH buffer, pH 8.0, 20 mM
MgCl2, 20 mM NaHCO3 (labeled with
14C to 2000 cpm nmol1 in the case of
14C assays), 0.1 mg mL1 BSA, 0.16 to 0.56 µg mL1 spinach Rubisco, and 250 to 500 µM
ribulose-P2. For spectrophotometric assays at 340 nm, based
on the procedure of Lilley and Walker (1974), the following coupling
enzymes and substrates were also included: 4 units mL1 of
yeast 3-phosphoglycerate kinase, 4 units mL1 of rabbit
muscle glyceraldehyde-3-phosphate dehydrogenase, 10 units
mL1 of rabbit muscle triose-phosphate isomerase, 4 units
mL1 of rabbit muscle glycerol-phosphate dehydrogenase, 4 units mL1 of rabbit muscle creatine kinase, 0.1 mg
mL1 of bovine carbonic anhydrase, 0.2 mM
NADH, 1 mM ATP, and 5 mM phosphocreatine. All
components except ribulose-P2 were incubated for at least
10 min at 25°C before the ribulose-P2 preparation was
added to initiate catalysis. For the radiometric assays, formic acid
was added after various intervals to aliquots of the mixtures to a
final concentration of 10% (v/v). The mixture was dried at 80°C and
nonvolatile radioactivity was measured by scintillation spectrometry.
For both assay methods, data from the resultant time courses were
fitted to the following equation, which models an exponential decay of
activity with time (t) from an initial, higher-activity
form (Vf) to a final, lower-activity form
(vf) with a half-time
(t1/2) of ln 2/kobs
(Edmondson et al., 1990a):
|
(1)
|
Analytical Anion-Exchange Chromatography
The procedure was adapted from that described by Harpel et al.
(1993). A Mono-Q 5/5 column equilibrated with 10 mM
Hepps-NaOH buffer, pH 8.0, containing 10 mM sodium borate
and 50 mM NaCl was used to resolve 100 nmol or less of
[1-14C]ribulose-P2 or
[1-3H]ribulose-P2 from
impurities. Samples were diluted, if necessary, with 5 mM
Hepps-NaOH buffer, pH 8.0, containing 5 mM sodium borate immediately before application. Two different elution protocols, both
using very shallow NaCl gradients in the equilibration buffer, were
used as noted in the figures. Protocol A (analogous to that of Chen and
Hartman [1995]) commenced at 50 mM NaCl, proceeded to 75 mM at 6.3 mM column
volume1, to 125 mM at 1 mM column volume1, to 200 mM at 3.8 mM column
volume1, and finally to 500 mM at
60 mM column volume1. Protocol B
commenced at 50 mM NaCl, proceeded to 225 mM at
2.1 mM column volume1, and finally
to 500 mM at 55 mM column
volume1. For both protocols, the flow rate of
the eluant was 1 mL min1. Fractions, usually of
1-min duration, were collected and their radioactivity was measured
after addition of an equal volume of Ultima Gold XR scintillant. Larger
amounts of labeled ribulose-P2 (up to 1 µmol)
were chromatographed on a Hema-IEC BIO 1000 Q 10U column (4.6 × 250 mm, Alltech) using the same eluant but with a steeper separating
NaCl gradient (4 mM column volume1)
and a faster flow rate (2 mL min1).
| |
RESULTS |
A H2O2-Sensitive Product of
Ribulose-P2 Oxidation Is a Major Cause of Fallover in Vitro
We noticed that the rate and extent of the slow inactivation
(fallover) of higher-plant Rubisco observed during extended assays varied between different preparations of
ribulose-P2 (data not shown). Preparations
purified by anion-exchange chromatography with shallow NaCl gradients
(see ``Materials and Methods'') showed the least fallover and did not
appear to deteriorate in this respect if stored at pH 2.8 in liquid
N2, even for periods as long as years. Storage of
ribulose-P2 preparations at a more neutral pH or
at 80°C or even a single additional freeze-thaw cycle caused
fallover to be noticeably more rapid and extensive. The deterioration
was particularly rapid if ribulose-P2 was stored
at room temperature in buffers usually used for Rubisco assays. Effects
became noticeable within 2 h of storage and worsened steadily
thereafter. The initial rates of Rubisco assays were reduced, the
deceleration occurred more rapidly, and the final, steady-state
activity eventually attained was strongly and progressively suppressed
as the period of storage of ribulose-P2
lengthened (Fig. 1A).
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| Figure 1.
Effect of storage of ribulose-P2 and
treatment with H2O2 on the time courses of
Rubisco activity assays. Assays were conducted as described in
``Materials and Methods'' using either the spectrophotometric (A and
B) or the radiometric (C and D) procedure. Ribulose-P2 was
derived from a freshly thawed aliquot that had been stored in liquid
N2 since synthesis. It was either used without further
treatment or preincubated aseptically in 125 mM Hepps-NaOH
buffer, pH 8.0, containing 25 mM MgCl2, at
22°C before use. A, Effect of increasing periods of preincubation:  , 0 min; , 130 min; - - -, 240 min; - , 480 min; and
- - , 1445 min. B, Effect of exclusion of O2 during
ribulose-P2 storage and/or Rubisco assay: , fresh ribulose-P2/aerobic assay; - - -, fresh
ribulose-P2/anaerobic assay; - - ,
ribulose-P2 stored aerobically for 24 h/aerobic assay; and
, ribulose-P2 stored anaerobically for 27 h in Mg2+-free buffer in the presence of Chelex 100 resin
(100-200 mesh, Na+ form, Bio-Rad)/anaerobic assay. C,
Reversal of inhibition by exposure of the stored
ribulose-P2 preparation to H2O2.
Ribulose-P2 was used without storage ( ), stored for
24 h ( ), or stored for 24 h followed by treatment with 1 M H2O2 for 30 min and removal of
H2O2 with 500 units of bovine catalase ( ).
D, Effect of H2O2 when present during assay of
Rubisco using fresh ribulose-P2: , no
H2O2; , 2 mM
H2O2;  , 4 mM
H2O2; and  , 6 mM
H2O2.
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Whereas the presence or absence of O2 during the
assay itself had no effect on the time course, the exclusion of
O2 and metals from the solution in which
ribulose-P2 was stored prevented the deterioration. Exclusion of either O2 or metals
was partially effective (data not shown), and when both were excluded
simultaneously, the time course showed slightly less deceleration even
than the control with freshly thawed ribulose-P2
(Fig. 1B). Fallover induced by stored ribulose-P2
was also ameliorated by treatment of the preparation with molar
concentrations of H2O2
before addition to the assay mixture (Fig. 1B). We confirmed earlier
observations (Badger et al., 1980; Edmondson et al., 1990a) that
millimolar concentrations of
H2O2 reduced the decline in
Rubisco activity if present in the assay mixture, although such
concentrations also inhibit the initial activity (Fig. 1D). These lower
H2O2 concentrations have
little or no ameliorating effect if used as a
ribulose-P2 pretreatment (data not shown).
The degree of fallover depended on the buffer in which
ribulose-P2 was stored (Table
I). It was worst when the storage
solution was buffered with Hepps, Hepes, or Mes. However,
ribulose-P2 stored in Tris, Tricine, Gly, or
triethanolamine buffers showed little or no enhancement of fallover
compared with controls with freshly thawed
ribulose-P2. Bicine buffer gave intermediate
results. Varying pH between 6.0 and 8.0 had little effect, strong
inhibition being seen with Mes at pH 6.0 and Hepps and Hepes at pH 8.0. Storage of ribulose-P2 in unbuffered solution was
strongly inhibitory at pH 8.2 and 6.3 (giving results similar to or
worse than Hepps, Hepes, or Mes at the same pH), less inhibitory at pH
4.0, and not inhibitory at all at pH 2.8. Significantly, storage in a
buffer mixture containing both Tris and Hepes caused little enhancement of fallover and the enhancement caused by storage in Hepps was substantially reversed by subsequent storage in Tris. However, the
reversing effect of Tris was not instantaneous because substitution of
Tris for Hepps in the assay buffer caused little reversal of the
enhancement of fallover caused by storage of
ribulose-P2 in Hepps (data not shown).
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Table I.
Influence of the buffer used for storage of
ribulose-P2 on the slow inhibition observed in subsequent
Rubisco activity assays
Ribulose-P2 was stored at room temperature in aseptically
filtered solutions with the stated components for the times shown and
then used to initiate Rubisco activity assays according to the
spectrophotometric method described in ``Materials and Methods''. The
time-course data were then fitted to Equation 1 to estimate the
parameters shown.
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Pi was released from ribulose-P2 during storage
at room temperature at pH 6.3 and 8.0 at rates approximating 0.1% per
hour regardless of the buffer present. Pi release was suppressed to barely detectable levels at pH 4.0 and below. However, there was no
correlation between the amount of Pi in a stored
ribulose-P2 preparation and the degree of
fallover it induced (data not shown).
A Tight-Binding Impurity Accumulates during Storage of
Ribulose-P2 at 80°C
Storage of
[1-14C]ribulose-P2
preparations at pH 2.8 and 80°C for extended periods led to the
formation of several impurities that could be resolved from
ribulose-P2 by anion-exchange chromatography (Fig. 2B). The most abundant of these,
designated "X," eluted higher in the NaCl gradient than
ribulose-P2 itself, suggesting that it contained
at least two phosphate moieties. When such preparations were allowed to
react to completion with fully activated spinach Rubisco, a residual
fraction of the radioactivity remained bound to the enzyme and could be
isolated by gel filtration (Fig. 2A). The binding was quite tight, with
no sign of release of labeled material from the trailing side of the
high-Mr peak. In this experiment, 8% of
the radioactivity in the starting ribulose-P2
preparation remained bound to Rubisco, and this was equivalent to 13%
of the Rubisco active sites present. The bound radioactivity released upon denaturation of the protein with SDS contained no
ribulose-P2, but most of the impurities observed
in the starting preparation were present (Fig. 2C). X predominated to a
greater degree among these than it did in the starting preparation, and
it appeared that X might be the tight-binding impurity, with the other
impurities being derived from it after release from the enzyme. This
interpretation was supported by the approximate agreement between the
amount of X measured by anion-exchange chromatography (6.3% of the
total radioactivity in Fig. 2B) and that observed bound to Rubisco
(8%, Fig. 2A). In a similar experiment with another labeled
ribulose-P2 preparation that contained a barely
detectable amount of X (< 1%), the
high-Mr complex isolated by gel filtration
contained only 0.7% of the total radioactivity (data not shown). From
this correspondence, we conclude that the bound material is derived from the starting preparation and is not produced in significant amounts as a by-product of catalysis at saturating
CO2.
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| Figure 2.
An impurity in a
14C-ribulose-P2 preparation that binds tightly
to Rubisco. A, [1-14C]ribulose-P2
(freshly thawed after 4 years of storage at pH 2.8 and at 80°C,
final concentration 30 µM, 82,000 cpm
nmol1) was mixed with preactivated spinach Rubisco (final
concentration 17 µM) in 45 mM Hepps-NaOH
buffer, pH 8.0, containing 13 mM MgCl2, 1 mM EDTA, and 9 mM NaHCO3. After 10 min at 22°C, 0.56 mL of this solution was applied to a 1- × 26-cm column of Sephadex G-50 (fine) equilibrated with the same buffer
components at a flow rate of 0.63 mL min1. The effluent
was monitored for radioactivity and A280. B,
Anion-exchange chromatography on a Mono-Q 5/5 column (see "Materials
and Methods," elution protocol A) of the
[1-14C]ribulose-P2 preparation used in A. C,
Fractions comprising the high-Mr peak shown
in A were pooled and SDS was added to 1% (w/v). Protein was removed by
ultrafiltration and an aliquot of the filtrate was chromatographed as
in B, approximately 2 h after the addition of SDS.
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Studies with X isolated chromatographically from stored labeled
ribulose-P2 preparations revealed that it was
unstable and supported the idea that the other impurities were derived
from it (Fig. 3). Rechromatography of the
isolated material after overnight storage in liquid
N2 revealed not only a predominant peak of X but
also the other impurities present in the starting
ribulose-P2 preparation (Fig. 3B). In addition to
degradation during storage and the associated freeze-thaw cycle, X
appeared to be degrading while bound to the anion-exchange column,
giving rise to diffuse peaks of radioactivity eluting before X itself.
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| Figure 3.
Characterization of the tight-binding impurity in
[1-14C]ribulose-P2. A, Twenty-six nanomoles
of [1-14C]ribulose-P2 (82,000 cpm
nmol1) was chromatographed on a Mono-Q 5/5 column as
described in ``Materials and Methods'' (elution protocol B).
Fractions comprising the 52-min peak were pooled and divided into
500-µL aliquots, each of which was subjected to one of the following
treatments and then rechromatographed. B, An aliquot was snap frozen,
stored overnight in liquid N2, diluted to 1 mL with
column-starting buffer, and rechromatographed. C, An aliquot was
diluted to 1 mL with 50 mM Hepps-NaOH buffer, pH 8.0, supplemented with o-phenylenediamine to 100 mM, and rechromatographed after storage for 1 h in the
dark at 22°C. D, To another aliquot, H2O2 was
added to a final concentration of 1.1 M. After 1 h at room temperature, water was added to 2 mL and 2,600 units of bovine catalase was added. Thirty minutes later, the mixture was snap frozen
and stored overnight in liquid N2 before rechromatography. The resulting chromatogram was aligned against a separate chromatogram of a 6:1 mixture of [3H]-P-glycolate and
[3H]-P-glycerate generated from
[1-3H]ribulose-P2 with R. rubrum Rubisco in a solution equilibrated with 500 µL
L1 CO2 in O2 as described by Kane
et al. (1994) but omitting the phosphatase treatment. The larger peak
of [3H]P-glycolate, eluting near 28 min and clearly
resolved from the closely preceding [3H]P-glycerate peak,
aligns precisely with the 28-min peak derived from the impurity. The
peaks eluting near 4 min are presumably the products of phosphatase
contamination of the enzyme preparations.
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Guessing that X might be pentodiulose-P2 produced
by oxidation of ribulose-P2 during storage
(Scheme 1), we reacted isolated X with
o-phenylenediamine, which converts vicinal dicarbonyl
compounds to 2,3-substituted quinoxalines. This had two effects on the
chromatographic behavior of X: it eluted slightly earlier in the NaCl
gradient (consistent with the observations of Chen and Hartman
[1995]) and the diffuse, early eluting peaks were suppressed (Fig.
3C).
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| Scheme 1.
Transition-metal-catalyzed oxidation of
ribulose-P2 and nonenzymatic cleavage of the resultant
pentodiulose-P2 by high concentrations of
H2O2. The asterisks indicate the fate of the
C-1 carbon of ribulose-P2.
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To confirm that X was indeed pentodiulose-P2, we
exposed it to 1.1 M
H2O2 and then catalase. A
very clean chromatogram resulted, showing only a major peak of
P-glycolate and a minor peak not retarded by the column that presumably
was glycolate resulting from phosphatase contamination in the catalase
preparation (Fig. 3D). The other product of
H2O2 cleavage of
pentodiulose-P2 would be P-glycerate derived from
carbons 3 to 5. This would not be labeled, since the radioisotope was
originally in the carbon 1 position of
ribulose-P2 (Scheme 1).
Pentodiulose-P2 Is Produced by Oxidation of
Ribulose-P2 with Cu2+
Fresh preparations of
[1-3H]ribulose-P2 or
those stored in liquid N2 contained no detectable
pentodiulose-P2 (Fig.
4, dotted line), but exposure of these
preparations to millimolar concentrations of Cu2+
at pH 8.0 for 3.5 h produced pentodiulose-P2
(3.5% of starting radioactivity) and other early eluting compounds
(Fig. 4, solid line). Inclusion of borate, which stabilizes
pentodiulose-P2 to some extent (Chen and Hartman,
1995) during the Cu2+ oxidation process, did not
increase the amount of pentodiulose-P2 recovered
(data not shown), perhaps because borate also forms a complex
with ribulose-P2 and therefore retards the
oxidation. Column fractions in the vicinity of the
pentodiulose-P2 peak were inhibitory when
preincubated with fully activated spinach Rubisco before assay, and the
peak of inhibition corresponded to the peak of 3H
radioactivity (Fig. 4). Inhibition increased as the concentration of
pentodiulose-P2 in the Rubisco assay increased
but it plateaued at approximately 75% inhibition (Fig. 4, inset). This
negatively cooperative pattern of inhibition is reminiscent of other
tight-binding inhibitors of Rubisco, such as carboxyarabinitol-1-P
(Gutteridge et al., 1986; Berry et al., 1987). Since the
Kd appeared to be close to the
concentration of Rubisco active sites present in the inhibition assays
(35 nM), the data were fitted to an equation that modeled
tight-binding inhibition (Fig. 4, inset). This estimated the
Kd to be 130 nM. However, the
negatively cooperative nature of the binding caused the data to fit the
model quite poorly. Clearly, the binding affinity is tighter than this
average estimate when site occupancy is low and looser when occupancy
is high. Since 75 mM NaCl and 4 mM sodium
borate were carried into the assay with the aliquots of column
fractions, this estimate must be regarded as a maximum estimate.
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| Figure 4.
Inhibition of Rubisco caused by a product of
ribulose-P2 oxidation.
[1-3H]ribulose-P2 (280 cpm
nmol1) was mixed with an air-equilibrated solution
containing 100 mM Hepps-NaOH, pH 8.0, and 2 mM
CuSO4 and stored at room temperature for 3.5 h. A 1-mL
sample was then applied to a 0.5-g column of Chelex 100 resin, and the
column was washed with 0.4 mL of 10 mM Hepps-NaOH buffer,
pH 8.0, containing 10 mM sodium borate. The eluant was
applied to the Hema-IEC column and chromatographed as described in
``Materials and Methods''. Fractions (1 min) were collected and 200 µL was removed for counting 3H (). Fresh
[1-3H]ribulose-P2, before Cu2+
treatment, was also chromatographed similarly (·······).
Fractions in the vicinity of the 58-min, peak of the chromatogram of
the Cu2+-treated sample were assayed within 10 min of
collection for ability to inhibit Rubisco (). A nonradioactive
fraction eluting between ribulose-P2 and X was used as the
control. Two-hundred microliters of each fraction was mixed with 200 µL of a solution of preactivated spinach Rubisco. After 5 min at
25°C, the reaction was initiated by a single addition of 75 µL of a
mixture of ribulose-P2 and NaH14CO3. The final concentrations of
components were: Rubisco, 2.3 µg mL1; Hepps-NaOH
buffer, pH 8.0, 90 mM; MgCl2, 17 mM; Na14HCO3 (4200 cpm
nmol1), 9.7 mM; NaCl, 75 mM;
sodium borate, 4 mM; ribulose-P2, 525 µM. After 2 min, the reaction was stopped by addition of
formic acid to 10% (v/v) and the mixtures were evaporated to dryness before addition of scintillant. 14C was determined by
scintillation spectrometry using a window that discriminated completely
against 3H. Inset, Plot of the extent of inhibition as a
function of the concentration of inhibitor present in the assays,
calculated from the 3H content. The dotted line shows the
best fit of the data to a rectangular hyperbola. The solid line shows
the best fit to the following equation, which models tight-binding
inhibition (adapted from Berry et al. [1987]):
|
(2)
|
where Et and
It are the total (bound plus free)
concentrations of Rubisco active sites and the inhibitor, respectively,
and Kd is the dissociation constant.
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The behavior of pentodiulose-P2 produced by
deliberate oxidation of
[1-3H]ribulose-P2 with
Cu2+ was identical to that of the inhibitor in
stored ribulose-P2 preparations. It bound tightly
to Rubisco to form a complex that could be isolated by gel filtration
in a manner similar to that shown in Figure 2A. The radioactivity
released from this high-Mr complex by
addition of SDS chromatographed predominantly in the X peak (analogous
to Fig. 2C). Alternatively, the label could be released by exposure of
the complex to 5 mM
H2O2, where P-glycolate and
a trace of nonphosphorylated material were the only products (analogous
to Fig. 3D). Cleavage of pentodiulose-P2 while it
was bound to the Rubisco active site was particularly facile.
Five-millimolar H2O2
effected complete cleavage in 30 min at 22°C, and this accords with
the effectiveness of such low concentrations in ameliorating the
deceleration of Rubisco assay time courses (Fig. 1D).
H2O2 concentrations 2 to 3 orders of magnitude higher are required to cleave
pentodiulose-P2 when it is free in solution
(Figs. 1C and 3D); therefore, we conclude that Rubisco must catalyze
the peroxidative cleavage of pentodiulose-P2
(Scheme 2).
View larger version (23K):
[in this window]
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| Scheme 2.
The structural analogy between the
ribulose-P2 enediol and pentodiulose-P2 when
bound within the active site of Rubisco, and the mechanistic analogy
between Rubisco-catalyzed oxygenation of the enediol and
Rubisco-catalyzed peroxidation of pentodiulose-P2 in the
presence of low concentrations of H2O2. R = -CHOH-CH2OPO32.
|
|
| |
DISCUSSION |
Pentodiulose-P2 Is Produced by Nonenzymatic Oxidation
of Ribulose-P2
Our data establish that pentodiulose-P2 can
be produced nonenzymatically from ribulose-P2 in
a reaction that depends on, or is accelerated by,
O2 and Chelex-removable metals (Scheme 1). It is
possible that transition metals are essential to the oxidation process
and are reoxidized by O2. If so,
pentodiulose-P2 might still be produced in the
absence of O2 in amounts stoichiometric with the
transition metals present. Although small, such amounts are still
likely to be significant compared with the Rubisco concentrations present in activity assays and, therefore, inhibitory. Pretreatment of
assay buffer components with Chelex would be only partially effective
in removing metal contamination because Mg2+,
which cannot be treated with Chelex, is required for activity and is a
likely source of the contamination. Deliberate oxidation of
ribulose-P2 with Cu2+
provides a ready means of synthesizing
pentodiulose-P2.
The identity of the oxidation product as
pentodiulose-P2 was established by its
chromatographic behavior and that of its adduct with
o-phenylenediamine, and the conversion of label present at C-1 to P-glycolate by cleavage with
H2O2 (Figs. 2-4).
Consistent with this identification, the mass spectrum (electron-impact
ionization) of the silyl derivative of the quinoxaline adduct formed
between o-phenylenediamine and the oxidation product
showed a prominent molecular ion at m/z = 436, together
with the expected m-15 and m-90 fragment ions resulting from losses of
a methyl group and trimethylsilanol, respectively (H.J. Kane and T.J.
Andrews, unpublished results). The oxidation product appears to be
identical in all of its properties to
pentodiulose-P2, previously identified as a
by-product of oxygenase catalysis by mutant (but not wild-type) R. rubrum Rubisco (Chen and Hartman, 1995; Harpel et al.,
1995b).
The rate of the oxidation reaction that proceeds without deliberate
addition of transition metals was not strongly dependent on pH between
8.3 and 6.0 but fell to insignificant levels at pH 2.8 (Table I). This
might indicate the involvement of the enediol form of
ribulose-P2 in the oxidation. Enediol formation by intramolecular abstraction (Richard, 1984) of the C-3 proton of
ribulose-P2 by O atoms of the phosphate groups
will only occur when the latter are unprotonated. However, whereas
inhibitor production at low pH was insignificant on a time scale of
days, significant amounts of the inhibitor accumulated at low pH during
protracted frozen storage at 80°C (Fig. 2). Perhaps the very slow
rate of inhibitor production under these conditions is offset by its
greater stability.
Pentodiulose-P2 Is an Excellent Analog of the
Enediol Intermediate
Pentodiulose-P2 bound very tightly to
carbamylated spinach Rubisco, forming a complex that could be isolated
by gel filtration (Fig. 2). We suggest that this strong binding
affinity is a result of the close structural resemblance between the
diulose-P2 and the enediol form of
ribulose-P2 (Scheme 2). All of the heavy atoms of
pentodiulose-P2 can adopt the same positions as
those of the enediol, and the planar configuration of C-4, C-3, O-3,
C-2, O-2, and C-1 can be emulated.
Since the complex can be isolated by gel filtration, taking tens of
minutes to hours without any sign of leakage of label from the trailing
side of the peak (Fig. 2), pentodiulose-P2 must not be prone to further conversion on the active site to other, less
tightly binding products. In this respect, the spinach enzyme differs
markedly from the R. rubrum enzyme. Wild-type R. rubrum Rubisco and its K329A mutant both convert
pentodiulose-P2 to
carboxytetritol-P2 (Harpel et al., 1995a,
1995b). This rearrangement product did not appear to remain tightly
bound to the K329A enzyme, since denaturation of the protein was not
required to recover it (Harpel et al., 1995b). By contrast, the E48Q
mutant of R. rubrum Rubisco apparently catalyzed the
O2-dependent cleavage of
pentodiulose-P2 to P-glycolate and P-glycerate
(Chen and Hartman, 1995). The inability of higher-plant Rubiscos to
catalyze similar transformations of pentodiulose-P2 to less tightly binding products
may be a reason for their susceptibility to fallover. Most bacterial
and algal Rubiscos show little sign of fallover (Gibson and Tabita,
1979; Andrews and Ballment, 1984; Yokota and Kitaoka, 1989; Lee at al., 1993; Hernandez et al., 1996), and we suggest that this might be
because they resemble the R. rubrum enzyme in being able to convert pentodiulose-P2 to quickly released
products.
We do not know whether, like ribulose-P2 and
xylulose-P2 (Jordan and Chollet, 1983; Zhu and
Jensen, 1991a), pentodiulose-P2 can also bind
tightly to the uncarbamylated active site. Fallover is not accompanied
by significant decarbamylation above pH 8.0 (Edmondson et al., 1990b;
Zhu and Jensen, 1991b). If pentodiulose-P2 is a
major cause of fallover, as our data appear to indicate, then this
inhibitor may resemble carboxyarabinitol-1-P in its preference for the
carbamylated active site (Seemann et al., 1985).
Pentodiulose-P2 Is Unstable
Pentodiulose-P2 decays to a variety of other
compounds. The chromatographic profiles (Figs. 2-4) indicate that
compounds with zero, one, and two phosphate groups are present among
the products, sometimes in multiple ionic forms within each class. The
label eluting higher in the NaCl gradient than
pentodiulose-P2 (e.g. the 82-min peak in Fig. 2,
B and C) may represent the product of the benzylic acid-type
rearrangement, carboxytetritol-P2. Plausible decay pathways resulting in the successive elimination of both phosphate groups of pentodiulose-P2 may also be
imagined. The evident diversity of decay products makes identifying all
of them a large task and we did not attempt it. Chen and Hartman (1995) and Harpel et al. (1995a) also observed extensive decay of
pentodiulose-P2 and showed that it could be
slowed by complexing with borate. We also used this strategy wherever
possible but found that it did not prevent the decay completely. In our
studies pentodiulose-P2 never accumulated to an
amount greater than 10% of that of the ribulose-P2 initially present. Apparently, a
steady state was attained in which the rate of appearance of
pentodiulose-P2 was balanced by the rate of its
further decay. Although the presence of borate slowed the rate of
decay, it slowed the oxidation leading to
pentodiulose-P2 even more so that the
steady-state amount of pentodiulose-P2 was
reduced when borate was included during the oxidation process (data not
shown).
The differences in severity of fallover induced by storage of
ribulose-P2 in different buffers (Table I) seem
most consistent with differences in the stability of
pentodiulose-P2 in different buffers. Although it
is possible that different degrees of contamination of the buffers with
transition metals could give rise to differences in the amount of
pentodiulose-P2 produced, serious fallover
occurred even when ribulose-P2 was stored without
buffer. Furthermore, buffers such as Tris were able slowly to reverse
the fallover-promoting tendency after it had been induced by storage of
ribulose-P2 in another buffer (Table I). This
points to a role of buffers such as Tris, Tricine, Gly, and
triethanolamine in accelerating the conversion of
pentodiulose-P2 to less-inhibitory compounds. The worst fallover was induced after ribulose-P2 had
been stored without buffer or in tertiary amine buffers. Primary and
secondary amine buffers were among those inducing the least fallover.
This might suggest a role for imines or enamines in the conversion were
it not for the discordance with this pattern of the tertiary amine triethanolamine, which induces very little fallover (Table I). However,
this discordance might be explained by the presence of approximately
1% mono- and diethanolamine in the reagent grade triethanolamine that
we used. Another tertiary amine, Bicine, had intermediate
fallover-inducing ability. Since diethanolamine is used in its
synthesis (Good et al., 1966), Bicine may also contain traces of
primary or secondary amines, explaining its intermediate status.
Reinterpretation of Earlier Observations about Fallover in the
Light of Pentodiulose-P2
Our observations that pentodiulose-P2 can be
a dominant cause of fallover, particularly with
ribulose-P2 of indifferent quality, demand that
previous explanations of fallover in terms of catalytic by-products be
reconsidered. Pentodiulose-P2 resembles the
fallover inhibitor(s) isolated from Rubisco reaction mixtures after
complete consumption of ribulose-P2 in several
respects. It binds to carbamylated Rubisco with similar affinity and
its binding shows the same negative cooperativity. Furthermore, it is
similarly unstable (Edmondson et al., 1990c). Edmondson et al. (1990d)
showed that two inhibitory compounds were present in their
preparations. One was clearly xylulose-P2 because
it was destroyed by aldolase and it produced xylitol and arabinitol
after reduction and dephosphorylation. They did not identify the other
conclusively, but showed that it was resistant to aldolase but
destroyed by brief exposure to mild alkali. Since the second inhibitor
appeared to produce predominantly arabinitol on reduction and
dephosphorylation, they speculated that it might be
ketoarabinitol-P2, produced by misprotonation of
the enediol form of ribulose-P2 at C-2. Zhu and
Jensen (1990b) appeared to substantiate this speculation by detecting
an inhibitor bound to Rubisco after catalysis that produced
predominantly arabinitol-1,5-bisphosphate on reduction.
However, the observations of Lee et al. (1993) and Chen and Hartman
(1995) that under some conditions borohydride reduction of
pentodiulose-P2 can produce predominantly
arabinitol-1,5-bisphosphate sound a note of caution about this
interpretation. Although such reduction should produce the
bisphosphates of ribitol, arabinitol, and xylitol in 1:2:1 proportions
if it was stereochemically impartial, it must be concluded that there
can be a strong preference for the arabinitol product under some
conditions and, therefore, that evidence for the presence of
ketoarabinitol-P2 based on detection of
arabinitol-1,5-bisphosphate after reduction must be considered unreliable. In view of this and our present results, it appears that
the second inhibitor observed by Edmondson et al. (1990d) may well have
been pentodiulose-P2 present not as a catalytic by-product but by virtue of its pre-existence in the starting ribulose-P2 preparation. Its alkali lability is
then readily explained by the enhancement of the benzylic acid-type
rearrangement of pentodiulose-P2 expected under
alkaline conditions. This reasoning, if correct, also implies that the
product of the rearrangement, carboxytetritol-P2,
is not a strong inhibitor of spinach Rubisco.
Another factor may have contributed to causing Edmondson et al.
(1990d) to overlook pentodiulose-P2. They used Tris buffer during gel filtration of Rubisco-bound inhibitors and subsequent work-up. This may have caused the decay of much of the
pentodiulose-P2 before reduction.
The relative contributions of pentodiulose-P2 and
xylulose-P2 (and
ketoarabinitol-P2, if it exits) to fallover is
now a moot point. The effect of pentodiulose-P2
clearly dominates when it is present in significant amounts in the
starting ribulose-P2. Even the reduced fallover
observed with the best ribulose-P2 preparations might not be caused solely by catalytic by-products, because storage of
ribulose-P2 in the absence of transition metals
and O2 caused slight further alleviation of
fallover (Fig. 1B). This may indicate that even our best preparations
of ribulose-P2 have traces of pentodiulose-P2 that disappear due to instability
when stored under conditions preventing further oxidation. Fallover
observed after this pretreatment was the slightest we have ever
observed (vf/vI = 0.65) and this residual level may represent the true contribution
of xylulose-P2 production. However, even in this circumstance, the possible contribution of traces of
pentodiulose-P2 still must not be overlooked.
Even in the absence of O2, traces of transition
metals introduced with the Mg2+ required for
assay might result in the production of stoichiometric amounts of
pentodiulose-P2 during the assay period itself.
Rubisco Catalyzes the Cleavage of Pentodiulose-P2
by H2O2
If pentodiulose-P2 emulates the enediol
intermediate in binding to Rubisco's active site, its facile cleavage
by low concentrations of
H2O2 can be easily
understood (Scheme 2). O2 addition to the enediol
and H2O2 addition to
pentodiulose-P2 are closely analogous and would
produce the same peroxyketone intermediate that would then be cleaved
to P-glycolate and P-glycerate by the normal oxygenase pathway.
H2O2-assisted release of
Rubisco from the otherwise dead-end complex with
pentodiulose-P2 provides a satisfying explanation for the long-standing observation that
H2O2 suppresses fallover (Badger et al., 1980). Earlier observations of an apparent lack of an
effect of H2O2 on the
isolated fallover inhibitor (Edmondson et al., 1990c) are now
explicable. The millimolar
H2O2 concentrations used in
that study are not effective in cleaving
pentodiulose-P2 when free in solution; molar
H2O2 concentrations are
required for this purpose. This relative resistance of unbound
pentodiulose-P2 to
H2O2 cleavage might be
expected if one of its keto groups was predominantly hydrated in
solution. Complexation with borate would also promote hydration. On the
active site, however, only the diketo form would emulate the enediol
intermediate. The instability of pentodiulose-P2
has so far frustrated attempts to chromatographically isolate it in
quantities sufficient for more detailed kinetic studies of its
Rubisco-catalyzed cleavage by
H2O2.
How Should Pentodiulose-P2 Formation Be Suppressed
during Synthesis and Storage of Ribulose-P2?
The need for ribulose-P2 preparations with
predictable and reproducible properties gives this question some
practical importance. Obviously, transition metals and
O2 should be excluded whenever possible. This can
be achieved easily enough during storage in liquid
N2 but it is more difficult during synthesis when
Mg2+, a potential source of other metals, must be
present. The use of shallow NaCl gradients to separate
pentodiulose-P2 from
ribulose-P2 during preparative anion-exchange
chromatography is therefore to be recommended. Storage at low pH
assists in suppressing the oxidation but it may also improve the
stability of the diulose-P2 product to some
extent.
Is Pentodiulose-P2 Produced from
Ribulose-P2 in Vivo?
Chloroplasts maintain high ribulose-P2
concentrations at around pH 8.0 during steady-state photosynthesis.
They are well supplied with O2 and the transition
metals required by the photosynthetic apparatus, such as Fe, Cu, and
others, must be present at finite concentrations. Under these
conditions it seems inevitable that pentodiulose-P2 must be formed and that it will
accumulate on Rubisco's active sites and inhibit photosynthesis
seriously unless specific mechanisms are present to prevent this from
happening. There have been reports of a tight-binding Rubisco inhibitor
in wheat and tobacco leaves that, unlike carboxyarabinitol-1-P, was present during the photoperiod but not in darkness (Keys et al., 1995;
Paul et al., 1996; Parry et al., 1997). It was detected in amounts
sufficient to inhibit 12% to 20% of the Rubisco present, shown to be
neither carboxyarabinitol-1-P nor xylulose-P2,
but otherwise not identified. Several of the properties of this daytime inhibitor (Keys at al., 1995; Parry et al., 1997) are suspiciously reminiscent of those of pentodiulose-P2 (this
study) and the fallover inhibitor (Edmondson et al., 1990c): (a) It
binds to Rubisco tightly enough to survive gel filtration but can be
released by dialyzing or gel filtering the complex in 200 mM SO42, whereupon
full recovery of the initial activity is obtained (Edmondson et al.,
1990c); (b) it elutes from anion-exchange columns higher in the NaCl
gradient than ribulose-P2; and (c) it is unstable at neutral pH and more stable in acidic conditions, and its inhibitory potency is diminished in Tris buffer. Reduction and dephosphorylation of the daytime inhibitor from wheat appeared to yield ribitol and
arabinitol, the same products yielded by
ribulose-P2 (Keys et al., 1995). In view of the
potential for stereochemical bias in the reduction of
pentodiulose-P2 discussed earlier, this
observation is not inconsistent with the inhibitor being
pentodiulose-P2. Further studies aimed at
identifying the daytime inhibitor are warranted. In particular, its
sensitivity to H2O2 needs
to be investigated.
Study of the physiological mechanisms that limit the accumulation of
pentodiulose-P2 in the chloroplast stroma is also
required. Two different classes of mechanisms might be present. First,
a mechanism for releasing Rubisco from its dead-end complex with pentodiulose-P2 must exist. Although cleavage of
the inhibitor by H2O2 on
the active site would accomplish this quite satisfactorily, it seems
that millimolar concentrations of
H2O2 are required for this
purpose (Fig. 1D; Badger et al., 1980). Chloroplasts have a very
effective ascorbate peroxidase mechanism for scavenging H2O2 that is thought to
keep the steady-state pool of
H2O2 below micromolar
concentrations (Asada, 1994). Therefore, sufficient H2O2 probably would not be
available for this release path to be feasible. Another release path
could be provided by Rubisco activase, which is known to facilitate the
release of a variety of inhibitors from both uncarbamylated and
carbamylated Rubisco (Portis, 1992; Salvucci and Ogren, 1996). Activase
is known to alleviate fallover in vitro (Robinson and Portis, 1989).
Second, if cleavage by H2O2
on Rubisco's active site is not possible, ancillary mechanisms for
detoxification and disposal of pentodiulose-P2
must exist. If the daytime inhibitor discussed in the preceding
paragraph is indeed pentodiulose-P2, then the small amounts detected must reflect a steady state between the rate of
formation by oxidation of ribulose-P2 and the
rate of disposal. A variety of possible disposal pathways may be
theorized. Dephosphorylation could occur, either before or after
rearrangement to carboxytetritol-P2 catalyzed
by glyoxylaselike enzymes. Alternatively, H2O2-dependent cleavage to
P-glycolate and P-glycerate catalyzed by a specific enzyme with a much
greater affinity for H2O2
than Rubisco would not only dispose of
pentodiulose-P2 safely but would also assist
ascorbate peroxidase in maintaining
H2O2 at a very low
concentration.
Our present data for spinach Rubisco support those of Chen and Hartman
(1995) for the wild-type R. rubrum enzyme in establishing that pentodiulose-P2 is not a significant
catalytic by-product when CO2 is saturating. This
is not surprising. Suppression of flux through the oxygenase catalytic
pathway by CO2 would minimize formation of
the peroxyketone intermediate (Scheme 2) from which pentodiulose-P2 might be derived by elimination
of H2O2. However, reports
that fallover of spinach Rubisco at pH 8.3 is exacerbated at
subsaturating CO2 (Edmondson et al., 1990a),
whereas decarbamylation is not (Edmondson et al., 1990b), raise
suspicions that some pentodiulose-P2 might be
produced enzymatically under these conditions. Further measurements of
pentodiulose-P2 (and
H2O2) production during
Rubisco catalysis at subsaturating CO2 are
required to address this issue. Any
pentodiulose-P2 produced by higher-plant Rubisco
under the physiologically relevant condition of
CO2 undersaturation would need to be released
from Rubisco and disposed of in the same manner as
pentodiulose-P2 produced by nonenzymatic
oxidation of ribulose-P2.
| |
FOOTNOTES |
1
This work was supported by the Australian
National University's Centre for Molecular Structure and Function.
2
Present address: Departement de Virologie,
Institut Pasteur Bruxelles, 642 rue Engeland, 1180 Bruxelles, Belgium.
*
Corresponding author; e-mail john.andrews{at}anu.edu.au; fax
61-2-6249-5075.
Received January 7, 1998;
accepted April 2, 1998.
| |
ABBREVIATIONS |
Abbreviations:
carboxyarabinitol-1-P, 2-carboxy-D-arabinitol-1-phosphate.
carboxytetritol-P2, 2-carboxytetritol-1,4-bisphosphate.
fallover, slow inactivation of Rubisco during catalysis.
ketoarabinitol-P2, 3-keto-D-arabinitol-1,5-bisphosphate.
P-glycerate, D-3-phosphoglycerate.
P-glycolate, 2-phosphoglycolate.
pentodiulose-P2, D-glycero-2,3-diulose-1,5-bisphosphate.
ribulose-P2, D-ribulose-1,5-bisphosphate.
xylulose-P2, D-xylulose-1,5-bisphosphate.
| |
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Abstract
Introduction