Interaction of ribulosebisphosphate carboxylase/oxygenase with transition-state analogs

Abstract

2-C-Carboxy-D-ribitol 1,5-bisphosphate and 2-C-carboxy-D-arabinitol 1,5-bisphosphate have been synthesized, purified, and characterized. In the presence of Mg2+, 2-C-carboxy-D-arabinitol 1,5-bisphosphate binds to ribulose-1,5-bisphosphate carboxylase/oxygenase by a two-step mechanism. The first, rapid step is similar to the binding of ribulose 1,5-bisphosphate or its structural analogues. The second step is a slower process (k = 0.04 s-1) and accounts for the tighter binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (Kd less than or approximately to 10(-11) M) than of 2-C-carboxy-D-ribitol 1,5-bisphosphate (Kd = 1.5 X 10(6) M). Both carboxypentitol bisphosphates exhibit competitive inhibition with respect to ribulose 1,5-bisphosphate. 2-C-(Hydroxymethyl)-D-ribitol 1,5-bisphosphate and 2-C-(hydroxymethyl)-D-arabinitol 1,5-bisphosphate were also synthesized; both are competitive inhibitors with respect to ribulose 1,5-bisphosphate with Ki = 8.0 X 10(-5) M and Ki = 5.0 X 10(-6) M, respectively. Thus, the carboxyl group of 2-C-carboxy-D-arabinitol 1,5-bisphosphate is necessary for maximal interaction with the enzyme. Additionally, Mg2+ is essential for the tight binding of 2-C-carboxy-D-arabinitol 1,5-bisophsphate. A model for catalysis of ribulose 1,5-bisphosphate carboxylation is discussed which includes a functional role for Mg2+ in the stabilization of the intermediate 2-C-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. Mechanistic implications that arise from the stereochemistry of this intermediate are also discussed.

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

“…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 carboxyarabinitol-P 2 , that binds stoichiometrically to CO 2 -Mg 21 activated Rubisco active sites almost irreversibly (Schloss, 1988) was purified from these preparations by ion exchange (Zhu and Jensen, 1991).…”

Ribulose Bisphosphate Carboxylase/Oxygenase Content Determined with [14C]Carboxypentitol Bisphosphate in Plants and Algae

“…Alternatively, Farquhar et al (12) and von Caemmerer and Farquhar (27) (26) and purified by chromatography (20). The prepared CPBP is an epimer mixture of CAPB and 2-carboxyribitol bisphosphate (20,22,26 25,000g for 15 min at 0WC, the resultant pellet and the centrifuge tube were extensively washed with 20% PEG-4000 containing the above solutes three times. The washed pellet was dissolved in 1 ml of 1% Triton using a Voltex mixter, and an aliquot of the Triton solution was used for determining radioactivity with a scintillation counter.…”

“…CPBP binds to Rubisco (9,20,22,26), and CABP, a structural analog of the transition state intermediate of the carboxylation reaction, binds with a Kd of 10-" M (22). Rather than precipitating the ['4C]CPBP-Rubisco complex with antibody (9), we describe in this paper a modification of the method using PEG-4000 (18) to precipitate the complex.…”

Characteristics of Light-Dependent Inorganic Carbon Uptake by Isolated Spinach Chloroplasts

“…After determining the acid labile-alkali stable "4C-radioactivity of each fraction, protein was precipitated with 5% (v/v) TCA and determined by the method of Lowry et al (17) using BSA as a standard. Unlabeled CPBP was prepared from NaCN and RuBP as described by Pierce et al (20).…”

“…Results of Figure 3 suggest that in addition to free HCO3-, some of the CO2 in the chloroplast is bound to the active site of RuBP carboxylase. Protein-carbamate was trapped by a method that takes advantage of the extremely stable, albeit slow forming complex, between 14C02-and Mg2`-activated RuBP carboxylase and a mixture of substrate analogs, CPBP, which are formed by a cyanohydrin reaction with RuBP (20). In this experiment, RuBP carboxylase was activated in situ with '4CO2 and the chloroplasts were osmotically ruptured in a medium designed to stabilize the enzyme-'4CO2-Mg2+-CPBP complex while minimizing the in vitro formation of protein-bound '4CO2.…”