©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Signature of the Oxygenase Intermediate in Catalysis by Ribulose-bisphosphate Carboxylase/Oxygenase as Provided by a Site-directed Mutant(*)

Yuh-Ru Chen (2), Fred C. Hartman (1)(§)

From the (1) Protein Engineering Program, Biology Division, Oak Ridge National Laboratory and the (2) University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37831

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Because of its inherent inefficiency and its dual activities of biosynthetic carboxylation and biodegradative oxygenation of RuBP, ()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.




EXPERIMENTAL PROCEDURES

Common laboratory reagents and biological materials were procured at the highest level of purity readily available. [1-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) .

Analyses of turnover products derived from [1-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).


RESULTS AND DISCUSSION

The active sites of Rubisco are interfacial (8) , being comprised of the /-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) .

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.


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 NHCl 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/VK 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).

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-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 HO 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 NHCl, 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.

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-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.

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 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

[1-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.



FOOTNOTES

*
This research was sponsored by the Office of Health and Environmental Research, United States Department of Energy, under Contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Biology Division, Oak Ridge National Laboratory, P. O. Box 2009, Oak Ridge, TN 37831-8077. Tel.: 615-574-0212; Fax: 615-574-9297.

The abbreviations used are: RuBP, D-ribulose 1,5-bisphosphate; PGA, 3-phospho-D-glycerate; PGyc, 2-phosphoglycolate; Rubisco, D-ribulose-1,5-bisphosphate carboxylase/oxygenase; XuBP, D-xylulose 1,5-bisphosphate; Bicine, N,N-bis(2-hydroxyethyl)glycine.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.