Potent Inhibition of Ribulose-Bisphosphate Carboxylase by an Oxidized Impurity in Ribulose-1,5-Bisphosphate1

Abstract

Oxidation of D-ribulose-1,5-bisphosphate (ribulose-P 2 ) during synthesis and/or storage produces D-glycero-2,3-pentodiulose-1,5-bisphosphate (pentodiulose-P 2 ), a potent slow, tight-binding inhibitor of spinach (Spinacia oleracea L.) ribulose-P 2 carboxylase/ oxygenase (Rubisco). Differing degrees of contamination with pentodiulose-P 2 caused the decline in Rubisco activity seen during Rubisco assay time courses to vary between different preparations of ribulose-P 2 . With some ribulose-P 2 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-P 2 did not appear to be a significant by-product of catalysis by wild-type Rubisco at saturating CO 2 concentration. It was produced slowly during frozen storage of ribulose-P 2 , even at low pH, more rapidly in Rubisco assay buffers at room temperature, and particularly rapidly on deliberate oxidation of ribulose-P 2 with Cu 2؉ . Its formation was prevented by the exclusion of transition metals and O 2 . Pentodiulose-P 2 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-P 2 by H 2 O 2 , producing P-glycolate.The photosynthetic CO 2 -fixing enzyme Rubisco catalyzes both carboxylation and oxygenation of ribulose-P 2 , 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-P 2 following abstraction of the proton attached to C-3 by an enzymatic base. This is followed by the attack of CO 2 or O 2 on C-2 of the resultant 2,3-enediol. Depending on whether the attacking species is CO 2 or O 2 , either 2Ј-carboxy-3-keto-arabinitol-1,5-bisphosphate or 2Ј-peroxy-3-keto-arabinitol-1,5-bisphosphate is formed as enzyme-bound intermediates. These intermediates are hydrated and cleaved between C-2 and C-3 to yield the products (for review, see Andrews and Lorimer, 1987; Hartman and Harpel, 1994;Gutteridge and Gatenby, 1995;Cleland et al., 1998).The activity of higher-plant Rubisco declines during assay in vitro. The decrease in activity is approximately exponential and commences as soon as fully activated Rubisco is mixed with ribulose-P 2 . Depending on the conditions, it proceeds with a half-time of 5 to 10 min and eventually reaches an apparent steady state in which the final activity is 20 to 50% of the initial activity. This phenomenon, now generally called "fallover," has been explained in terms of the production of isomers of ribulose-P 2 , xylulose-P 2 , and ketoarabinitol-P 2 by stereochemically incorrect reprotonation of the enediol intermediate (Edmondson et al., 1990a(Edmondson et al., , 1990b(Edm...

Isoleucine 309 acts as a C 4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

“…RuBP and [ 14 C]2-CABP were synthesized as described (31,32). Protein content was measured using a dye-binding assay (Pierce) and BSA as a proteinstandard.…”

The Catalytic Properties of Hybrid Rubisco Comprising Tobacco Small and Sunflower Large Subunits Mirror the Kinetically Equivalent Source Rubiscos and Can Support Tobacco Growth

“…Substrate ribulose-P 2 (both 1 3 H labeled and unlabeled) was synthesized according to Kane et al (1998). Unlabelled ([ 12 C]) and carboxyl-14 C-labeled ([ 14 C]) carboxypentitol-P 2 (an isomeric mixture of carboxyarabinitol-P 2 and carboxyribitol-P 2 ) were synthesized as described in Pierce et al (1980).…”

The Rubisco Chaperone BSD2 May Regulate Chloroplast Coverage in Maize Bundle Sheath Cells

“…Total Rubisco activity was determined after Rubisco in the leaf protein was activated for 10 min in assay buffer before initiating Rubisco activity measurements by adding RuBP to 0.4 mM. The RuBP used in the assays was synthesized and purified as described previously (Kane et al, 1998). Rubisco content was determined in 100 mL of soluble extract by [ 14 C]CABP binding (Sharwood et al, 2008).…”