Potent inhibition of ribulose-bisphosphate carboxylase by an oxidized impurity in ribulose-1,5-bisphosphate

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.

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 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; Δ, 4 mm H₂O₂; and ○, 6 mm H₂O₂.

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 nmol⁻¹) 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 min⁻¹. 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-M_r 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.

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 nmol⁻¹) was chromatographed on a Mono-Q 5/5 column as described in 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 L⁻¹ 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.

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.

Figure 4

Inhibition of Rubisco caused by a product of ribulose-P₂ oxidation. [1-³H]ribulose-P₂ (280 cpm nmol⁻¹) 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 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 mL⁻¹; Hepps-NaOH buffer, pH 8.0, 90 mm; MgCl₂, 17 mm; Na¹⁴HCO₃ (4200 cpm nmol⁻¹), 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 E_t and I_t are the total (bound plus free) concentrations of Rubisco active sites and the inhibitor, respectively, and K_d is the dissociation constant.

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⁻.