From the
An uncharacterized minor transient product, observed in our
earlier studies of substrate turnover by the E48Q mutant of
Rhodospirillum rubrum ribulose-bisphosphate
carboxylase/oxygenase (Lee, E. H., Harpel, M. R., Chen, Y.-R., and
Hartman, F. C.(1993) J. Biol. Chem. 268, 26583-26591), becomes
a major product when it is trapped and stabilized with borate as an
additive to the reaction mixture. Chemical characterization establishes
this novel product as D-glycero-2,3-pentodiulose
1,5-bisphosphate, thereby demonstrating oxidation of the C-3 hydroxyl
of D-ribulose 1,5-bisphosphate to a carbonyl. As the formation
of the novel oxidation product is oxygen-dependent and generates
hydrogen peroxide, its precursor must be a peroxy derivative of
ribulose bisphosphate. Thus, discovery of the dicarbonyl bisphosphate
lends direct support to the long standing, but heretofore unproven,
postulate that the normal pathway for oxidative cleavage of ribulose
bisphosphate by the wild-type enzyme entails a peroxy intermediate. Our
results also suggest that stabilization of the peroxy intermediate by
the wild-type enzyme promotes carbon-carbon scission as opposed to
elimination of hydrogen peroxide.
Because of its inherent inefficiency and its dual activities of
biosynthetic carboxylation and biodegradative oxygenation of
RuBP
Common laboratory reagents and biological materials were
procured at the highest level of purity readily available.
[1-
Analyses of turnover products derived from
[1-
The active sites of Rubisco are interfacial
(8) , being
comprised of the
We have used site-directed
mutagenesis to assess the roles of the intersubunit salt bridge and the
residues therein
(5, 11) , as well as loop 6 perse(12) , in intermediate stabilization and control
of ligand ingress to and egress from the active site. Prior
characterization
(5) of the E48Q mutant showed that Glu-48
enhances the carboxylation rate by 200-fold and that disruption of the
salt bridge decreases the carboxylation/oxygenation partitioning ratio
and leads to extensive misprotonation of the intermediate enediolate at
C-3 to form XuBP, a poor alternate substrate for Rubisco. Whereas this
error occurs only once per 400 turnovers with the native spinach enzyme
(13) and is undetectable with the wild-type R. rubrum enzyme
(5) , misprotonation and carboxylation of the
enediolate occur at similar rates with the E48Q mutant. In addition to
XuBP, an uncharacterized minor side product (denoted unknown in
Fig. 2
) was also formed during turnover of RuBP by E48Q.
Its late elution
position from the anion-exchange column, in conjunction with the oxygen
requirement for its formation, indicated that the unknown might be an
oxidized analogue of RuBP with both phosphate groups intact. A compound
fitting this description is
D-glycero-2,3-pentodiulose-1,5-bisphosphate. Such a
dicarbonyl bisphosphate should undergo oxidative cleavage by hydrogen
peroxide to form PGyc and PGA, condensation with
o-phenylenediamine to form a dioxime, and reduction with
sodium borohydride to form ribitol bisphosphate, arabinitol
bisphosphate, and xylitol bisphosphate in molar ratios of 1:2:1
(Fig. 3). Purified unknown (Fig. 4), derived from
[1-
The formation of PGA and PGyc
as the sole end products of RuBP turnover by E48Q and the
disproportionate increase in labeled PGyc (from
[1-
Oxidation of RuBP by molecular oxygen
to form a 2,3-pentodiulose can only occur via a C-2 peroxy intermediate
(Fig. 5), identical to that proposed in the normal oxygenase
pathway of Rubisco catalysis leading to PGA and PGyc
(3) .
Although this earlier postulate was chemically sound and was supported
by the appearance in PGyc of one atom of oxygen derived from molecular
oxygen and also supported by
[1-
, (
)D-ribulose-1,5-bisphosphate
carboxylase/oxygenase (EC 4.1.1.39) is viewed as a fulcrum of biomass
yields (see Ref. 1 for a review of Rubisco). Catalytic turnover of RuBP
by Rubisco involves several unstable intermediates (Fig. 1): the
2,3-enediolate of RuBP (I), 2-carboxyl-3-ketoarabinitol
1,5-bisphosphate (and its hydrate) (II, III), and the
C2 carbanion of PGA (VI) all in the carboxylation pathway and
the same enediolate (I) and a putative C-2 peroxy derivative
of RuBP (and its hydrate) (IV, V) in the oxygenation
pathway. Questions then arise as to how the enzyme mitigates
decomposition of labile intermediates and ensures normal throughput of
substrate. We are attempting to address these questions by use of
site-directed mutagenesis to probe potentially relevant structural
elements as suggested by the x-ray structure
(2) . One such
structural element is a mobile segment of the N-terminal
domain of the interfacial active site, which includes the catalytically
facilitative residue Glu-48. In this report, we show that the E48Q
mutant of the Rhodospirillum rubrum enzyme, although severely
impaired in carboxylase activity, catalyzes the oxidation of RuBP to
D-glycero-2,3-pentodiulose 1,5-bisphosphate. As a
signature of the corresponding peroxy derivative of RuBP, this novel
dicarbonyl product provides the most direct evidence to date for a
peroxy intermediate in the normal oxygenase pathway as first invoked in
pioneering studies of Lorimer et al.(3) .
Figure 1:
Reaction pathways for the carboxylation
and oxygenation of RuBP as catalyzed by
Rubisco.
H]RuBP and [5-
H]RuBP
were synthesized from commercial [2-
H]glucose and
[6-
H]glucose, respectively, by a published
procedure (4). The E48Q mutant of R. rubrum Rubisco was
isolated from transformed Escherichia coli MV1190 as described
earlier
(5) and was homogeneous, as judged by Coomassie Blue
staining of SDS-polyacrylamide gels. The concentration of the mutant
protein was based on the extinction coefficient at 280 nm of the
wild-type enzyme (A of 1.2 for 1 mg/ml)
(6) .
H]RuBP or [5-
H]RuBP
followed the published anion-exchange method (7). Detailed reaction
conditions are provided in the figure and table legends. Except where
noted, all reactions were carried out at room temperature and included
air-saturated concentrations of O
(255 µM).
/
-barrel domain from one subunit and the
N-terminal domain of the adjacent identical subunit (9).
During catalysis, the active sites are apparently occluded by flexible
segments (loop 6 from the
/
-barrel domain and helix B from
the N-terminal domain) that extend over the mouth of the
barrel and approach each other. In this closed conformation, visualized
directly in the three-dimensional structure of the exchange-resistant
complex of activated enzyme and the reaction-intermediate analogue
2-carboxyarabinitol 1,5-bisphosphate, active-site Lys-329 (located at
the apex of loop 6) and active-site Glu-48 (located at the
C-terminal end of helix B) engage in intersubunit
electrostatic interaction
(10) .
Figure 2:
Anion-exchange chromatographic analyses of
radioactive products generated from [1-H]RuBP by
E48Q. [1-
H]RuBP (250 µM) and E48Q (1
mg/ml) were incubated in 0.5 ml of pH 8.0 buffer (50 mM
Bicine, 66 mM NaHCO
, 10 mM
MgCl
, 1 mM EDTA, 1 mM 2-mercaptoethanol,
and 2% glycerol). At 30 s (panelA), 10 min
(panelB), and 60 min (panelC),
100-µl aliquots of the reaction mixture were quenched with 1%
(w/v) SDS, deproteinated (Amicon Centricon-10
filters), and chromatographed at pH 8.0 on a MonoQ anion exchanger
(Pharmacia Biotech Inc. HR5/5, 5
50 mm) with a gradient
(depicted only in panelA) of NH
Cl
containing a fixed concentration of 10 mM sodium borate.
Radioactivity was monitored continuously by flow-through radiometric
detection (IN/US
-RAM).
values were calculated from the
ratio of radioactive peak areas for PGA and PGyc (corresponding to the
kinetic partitioning between carboxylation and oxygenation) according
to the relationship,
=
(
/
)/([CO
]/[O
])
(14). PanelD depicts an identical, 60-min reaction
mixture (100 µl), except for the inclusion of 80 mM
borate. Analysis of a third reaction mixture (300 µl) (30 min),
which was identical to that shown in panelD but
freed of oxygen by inclusion of protocatechuate and protocatechuate
3,4-dioxygenase (7), is shown in panelE.
Unk, unknown.
Curiously, the carboxylation/oxygenation partitioning ratio
(VK
/V
K
denoted as
)
(14) declines during progression of
[1-
H]RuBP utilization by E48Q. For example, the
value provided by the
[3-
H]PGA/[2-
H]PGyc ratio
declines from 2.0 at early stages of RuBP utilization to 0.3 at
completion, with PGA and PGyc as the sole end products (Fig. 2,
A-C). By contrast, the
value of the
wild-type enzyme is about 11
(7, 15) and is independent
of the extent of RuBP turnover. Thus, a transient product (XuBP or the
unknown) must be partitioned differently than RuBP by the mutant
enzyme. Since XuBP and RuBP should yield the same enediolate, the
unknown presumably accounts for the
dependence on the extent of
RuBP turnover. This contention is supported by inclusion of borate in
the reaction mixture, thereby trapping the unknown and retarding its
subsequent processing (Fig. 2D). Under these conditions,
35% of the input RuBP is converted to the unknown, and the
value
is raised to 1.6 (i.e. disproportionately less PGyc is formed
when processing of the unknown is blocked). The unknown is not formed
in the absence of oxygen (Fig. 2E).
H]RuBP, was subjected to each of these
chemical treatments, and the resulting products were identified by
anion-exchange chromatography. As predicted, cleavage of the unknown
with hydrogen peroxide gave PGyc as the only labeled product
(Fig. 4B), condensation with o-phenylenediamine
gave complete conversion to a less acidic compound
(Fig. 4C), and borohydride reduction gave an approximate
1:2:1 mixture of the three pentitol bisphosphates
(Fig. 4D). These experiments were repeated with purified
unknown derived from [5-
H]RuBP. Chromatographic
profiles were identical to those shown in Fig. 4with the
exception of PGA rather than PGyc as the only labeled product of
oxidative cleavage (data not shown). We conclude that the unknown is
D-glycero-2,3-pentodiulose-1,5-bisphosphate and
stress that neither the corresponding 2,4-pentodiulose nor any of the
pentulose isomers would exhibit the observed chemical properties.
Figure 3:
Predicted reactions of
D-glycero-2,3-pentodiulose 1,5-bisphosphate with
hydrogen peroxide, o-phenylenediamine, or sodium
borohydride.
Figure 4:
Characterization of
D-glycero-2,3-pentodiulose 1,5-bisphosphate. The
dicarbonyl bisphosphate (55 µmol) was isolated by MonoQ
chromatography of a reaction mixture consisting of
[1-H]RuBP and E48Q as described in the legend for
Fig. 2D. Rechromatography on MonoQ of a portion of the
isolated material demonstrates its purity (panelA).
Another portion of the isolated material (7 µM) was
treated with 1 M hydrogen peroxide in 10 mM sodium
borate (pH 8.0) for 1 h. The reaction mixture was quenched with 9
volumes of H
O followed by the addition of 2,000 units of
catalase. Following deproteination, the sample was chromatographed
(panelB). A third portion of the dicarbonyl
bisphosphate (4 µM) in 50 mM Bicine (pH 8.0) was
incubated with 100 mMo-phenylenediamine for 1 h
prior to chromatography (panelC). A fourth portion
of the dicarbonyl bisphosphate (7 µM) was treated at 2
°C with 10 mM NaBH
for 15 min in a pH 8.0
buffer containing 50 mM Bicine, 85 mM NaCl, 120
mM NH
Cl, 10 mM MgCl
, 66
mM NaHCO
, and 1 mM EDTA and
chromatographed subsequent to the addition of glucose (20 mM)
to consume excess borohydride (panelD); peak
assignments are based on standards (7). A fifth portion of the
dicarbonyl bisphosphate (7.8 µM) was incubated with E48Q
(1 mg/ml) for 24 h in the pH 8.0 Bicine buffer described in the legend
for Fig. 2, quenched with 1% (w/v) SDS, deproteinated, and
chromatographed (panelE). Unk,
unknown.
In
our earlier study
(5) , we tentatively (and erroneously)
identified this compound as 3-ketoarabinitol 1,5-bisphosphate, which
would be formed by misprotonation of the enediolate at C-2 and has
indeed been reported as a minor product with wild-type spinach
Rubisco
(16) . Our misidentification was based on borohydride
reduction in which arabinitol 1,5-bisphosphate was the only significant
product. The reduction was carried out at pH 8.0 in 120 mM
NHCl containing 10 mM sodium borate, i.e. the solvent composition for the compound as eluted from the MonoQ
column. We have confirmed that reduction of
D-glycero-2,3-pentodiulose 1,5-bisphosphate under
these conditions yields arabinitol 1,5-bisphosphate almost exclusively.
In contrast, at higher ionic strengths as used in the present study and
described in the legend for Fig. 4D, the reduction leads
to the statistically predicted ratio of the three pentitol
bisphosphates. We conclude that at relatively low ionic strengths
complexation of borate by the dicarbonyl compound results in
stereoselective borohydride reduction.
H]RuBP as substrate) as turnover progresses
prompt the deductions that
D-glycero-2,3-pentodiulose-1,5-bisphosphate must
serve as an alternate substrate for the mutant enzyme and that PGyc
must be a product. Incubation of the isolated
[1-
H]dicarbonyl bisphosphate with E48Q indeed
gives PGyc as the only labeled product (Fig. 4E),
whereas turnover of the isolated [5-
H]dicarbonyl
bisphosphate identifies PGA as the other cleavage product (data not
shown). Although we have not studied the enzyme-catalyzed turnover of
the dicarbonyl bisphosphate in detail, the process is oxygen-dependent
(data not shown) and thus appears similar to oxidative cleavage of the
compound by hydrogen peroxide.
O-EPR
(17) , proof has
been lacking due to inherent instability of the putative intermediate.
Our discovery of D-glycero-2,3-pentodiulose
1,5-bisphosphate provides a direct signature of the oxygenase
intermediate and thus confirms the proposed pathway for oxidative
cleavage of RuBP by Rubisco. Our studies also illustrate alternate
fates for the peroxy intermediate: carbon-carbon scission as promoted
by wild-type enzyme or elimination of hydrogen peroxide as promoted by
E48Q. Formation of hydrogen peroxide concomitant with the dicarbonyl
bisphosphate has been verified by use of peroxidase and catalase
(). Thus, the demonstrated chemistry is reminiscent of
flavin hydroperoxides, which dissociate to hydrogen peroxide and
oxidized flavin (see Ref. 18 for a review).
Figure 5:
Formation of the C-2 peroxy compound from
RuBP and partitioning to D-glycero-2,3-pentodiulose
1,5-bisphosphate or PGyc and PGA.
Outstanding issues,
worthy of future consideration, include identification of factors that
dictate the ultimate fate of the dicarbonyl bisphosphate, the mechanism
of its enzyme-catalyzed oxidative cleavage, whether it might be formed
with wild-type enzyme but cleaved prior to dissociation from the active
site and hence never before detected, and whether it might be partially
responsible for the time-dependent inactivation of higher plant Rubisco
that occurs during RuBP
turnover
(13, 16, 19, 20) .
Table:
Formation of hydrogen peroxide during turnover
of RuBP by the E48Q mutant
H]RuBP (250
µM) and E48Q (1 mg/ml) were incubated in 300 µl of pH
8.0 Bicine buffer (inclusive of 80 mM sodium borate) as
described in the legend for Fig. 2D with and without catalase
(1000 units). After 1 h, one-third of each reaction mixture was mixed
with an equal volume of 2 M sodium acetate to lower the pH to
5.4 and then assayed for peroxide; the remainder of each reaction
mixture was quenched with 1% (w/v) SDS, deproteinated, and
chromatographed.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.