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.)
Oxidation of D-ribulose-1,5-bisphosphate (ribulose-P2) during
synthesis and/or storage produces D-glycero-2,3-pentodiulose-1,5bisphosphate (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 Cu21. 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.
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 29-carboxy-3-keto-arabinitol1,5-bisphosphate or 29-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
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 [email protected]; fax
61–2– 6249 –5075.
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 ribuloseP2, 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
Abbreviations: carboxyarabinitol-1-P, 29-carboxy-d-arabinitol-1phosphate; carboxytetritol-P2, 29-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.
1059
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1060
Kane et al.
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.
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 280°C for approximately 4 years before use. This
preparation contained impurities arising from decay of
ribulose-P2 during storage (see “Results”). [1-3H]RibuloseP2 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 volume21 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- 3 100-cm
column of AG1-X8 (Cl2 form, Bio-Rad) equilibrated with 3
mm HCl, using a linear NaCl gradient rising at 24 mm
column volume21 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-N9-3-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 solu-
Plant Physiol. Vol. 117, 1998
tions containing 100 mm Hepps-NaOH buffer, pH 8.0, 20
mm MgCl2, 20 mm NaHCO3 (labeled with 14C to 2000 cpm
nmol21 in the case of 14C assays), 0.1 mg mL21 BSA, 0.16 to
0.56 mg mL21 spinach Rubisco, and 250 to 500 mm ribuloseP2. 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
mL21 of yeast 3-phosphoglycerate kinase, 4 units mL21 of
rabbit muscle glyceraldehyde-3-phosphate dehydrogenase,
10 units mL21 of rabbit muscle triose-phosphate isomerase,
4 units mL21 of rabbit muscle glycerol-phosphate dehydrogenase, 4 units mL21 of rabbit muscle creatine kinase, 0.1
mg mL21 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):
product 5 Vf 3 t 1
~vi 2 vf!~1 2 ekobs 3 t!
kobs
(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 volume21, to 125 mm at 1 mm column volume21, to
200 mm at 3.8 mm column volume21, and finally to 500 mm
at 60 mm column volume21. Protocol B commenced at 50
mm NaCl, proceeded to 225 mm at 2.1 mm column volume21, and finally to 500 mm at 55 mm column volume21.
For both protocols, the flow rate of the eluant was 1 mL
min21. 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 mmol) were chromatographed on a Hema-IEC BIO 1000 Q 10U column
(4.6 3 250 mm, Alltech) using the same eluant but with a
steeper separating NaCl gradient (4 mm column volume21)
and a faster flow rate (2 mL min21).
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Inhibition of Rubisco by Oxidized Ribulose Bisphosphate
1061
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 280°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).
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 ribuloseP2. 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
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
Mg21-free buffer in the presence of Chelex 100 resin (100–200 mesh,
Na1 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 (F), stored for 24 h (ƒ), 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 (M). D, Effect of H2O2 when
present during assay of Rubisco using fresh ribulose-P2: F, no H2O2;
M, 2 mM H2O2; D, 4 mM H2O2; and E, 6 mM H2O2.
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1062
Kane et al.
Plant Physiol. Vol. 117, 1998
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.
Buffer Used for Storage
pH
Storage Time
h
Control (no storage)
No buffer
No buffera
No buffera
No buffera
Hepes-NaOH (100 mM)
Hepps-NaOH (100 mM)
Mes-NaOH (100 mM)
Bicine-NaOH (100 mM)
Glycine-NaOH (100 mM)
Tricine-NaOH (100 mM)
Tris-HCl (100 mM)
Tris-HCl (100 mM), MgCl2 (20 mM)
Triethanolamine-HCl (100 mM)
Hepes-NaOH (50 mM)
Hepes-NaOH (50 mM), Tris-HCl (50 mM)
Hepps-NaOH then Tris-HClb
—
2.8
4.0
6.3
8.2
8.0
8.0
6.0
8.0
8.0
8.0
8.0
8.0
8.3
8.0
8.0
8.0
—
18
24
24
30
18
18
18
18
18
46
18
18
20
4
4
18/18
vi
s
vf /vi
t1/2
0.39
0.41
0.26
0.07
0.06
0.06
0.07
0.15
0.22
0.37
0.38
0.41
0.40
0.39
0.21
0.26
0.49
15
15
7.8
1.2
0.8
2.2
3.0
2.7
6.9
11
11
14
11
11
2.5
9.6
7.0
21
2.9
2.7
2.4
2.5
0.9
1.3
1.2
2.3
2.3
2.8
2.9
2.7
2.7
2.9
2.2
2.7
2.1
min
a
b
pH adjusted with NaOH.
Ribulose-P2 was stored for 18 h in 100 mM Hepps-NaOH, then diluted 6-fold into 100 mM Tris-HCl and stored
for a further 18 h before addition to an assay containing 100 mM Tris-HCl, pH 8.0.
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).
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 280°C
Storage of [1-14C]ribulose-P2 preparations at pH 2.8 and
280°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 predomi-
nated 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.
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.
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
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Inhibition of Rubisco by Oxidized Ribulose Bisphosphate
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 280°C, final concentration 30 mM, 82,000
cpm nmol21) was mixed with preactivated spinach Rubisco (final
concentration 17 mM) 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- 3 26-cm column
of Sephadex G-50 (fine) equilibrated with the same buffer components at a flow rate of 0.63 mL min21. 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.
[1995]) and the diffuse, early eluting peaks were suppressed (Fig. 3C).
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
1063
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 nmol21) 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-mL 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 columnstarting 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 mL L21 CO2 in O2 as described by Kane et al. (1994) but
omitting the phosphatase treatment. The larger peak of [3H]Pglycolate, 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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1064
Kane et al.
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.
Plant Physiol. Vol. 117, 1998
nantly 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
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 Cu21
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 Cu21 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 Cu21 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.
The behavior of pentodiulose-P2 produced by deliberate
oxidation of [1-3H]ribulose-P2 with Cu21 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 predomi-
Figure 4. Inhibition of Rubisco caused by a product of ribulose-P2
oxidation. [1-3H]ribulose-P2 (280 cpm nmol21) 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 mL was
removed for counting 3H (——). Fresh [1-3H]ribulose-P2, before
Cu21 treatment, was also chromatographed similarly (zzzzzzz). Fractions
in the vicinity of the 58-min, peak of the chromatogram of the
Cu21-treated sample were assayed within 10 min of collection for
ability to inhibit Rubisco (E). A nonradioactive fraction eluting between ribulose-P2 and X was used as the control. Two-hundred
microliters of each fraction was mixed with 200 mL of a solution of
preactivated spinach Rubisco. After 5 min at 25°C, the reaction was
initiated by a single addition of 75 mL of a mixture of ribulose-P2 and
NaH14CO3. The final concentrations of components were: Rubisco,
2.3 mg mL21; Hepps-NaOH buffer, pH 8.0, 90 mM; MgCl2, 17 mM;
Na14HCO3 (4200 cpm nmol21), 9.7 mM; NaCl, 75 mM; sodium
borate, 4 mM; ribulose-P2, 525 mM. 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]):
inhibition % 5 100
H
Et 1 It 1 Kd 2
Î~Et 1 It 1 Kd!2 2 4 z It z Et
2 z Et
J
(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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Inhibition of Rubisco by Oxidized Ribulose Bisphosphate
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).
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, inhibi-
1065
tory. Pretreatment of assay buffer components with Chelex
would be only partially effective in removing metal contamination because Mg21, which cannot be treated with
Chelex, is required for activity and is a likely source of
the contamination. Deliberate oxidation of ribulose-P2
with Cu21 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 ophenylenediamine and the oxidation product showed a
prominent molecular ion at m/z 5 436, 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 byproduct of oxygenase catalysis by mutant (but not wildtype) 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 280°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
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 5
-CHOH-CH2OPO322.
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 rearrange-
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Copyright © 1998 American Society of Plant Biologists. All rights reserved.
1066
Kane et al.
ment 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
Plant Physiol. Vol. 117, 1998
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
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Inhibition of Rubisco by Oxidized Ribulose Bisphosphate
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 Rubiscobound 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 5 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 Mg21 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 concen-
1067
trations 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 Mg21, 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 SO422, 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
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1068
Kane et al.
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
Plant Physiol. Vol. 117, 1998
pentodiulose-P2 produced by nonenzymatic oxidation of
ribulose-P2.
Received January 7, 1998; accepted April 2, 1998.
Copyright Clearance Center: 0032–0889/98/117/1059/11.
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