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...

The CO2-fixing enzyme Rubisco plays a crucial biological role as a primary determinant of both plant yield and the response of the biosphere to global change. Here, we describe techniques for measuring the amount and activity of Rubisco in higher plants. To accommodate a range of experimental capabilities, we describe basic radioisotopic methods as well as non-radioactive techniques. The required calculations are included. We discuss problems that commonly arise during the extraction and assay of Rubisco.

The inhibition of purified spinach ribulo~bisphosphate carboxylase-oxygenase which occurs progressively during catalysis in vitro is caused by accumulation of at least two tight-binding inhibitors at the catalytic site. Reduction of these inhibitors with NaB3H,, followed by dephosphorylation, produced a mixture of xylitol and arabinitol, thus identifying one of them as D-xylulose 1,Sbisphosphate. It was formed during carboxylation, presumably by a stereochemically incorrect reprotonation of the 2,3-enediolate intermediate bound at the catalytic site. Under the conditions used, this epimerization occurred approximately once for every 400 carboxylation turnovers. Another inhibitor may be 3-keto-D-arabinitoi l$bisphosphate which would also be formed by misprotonation of the enediolate intermediate, but at C-2 rather than at C-3.

Methods for determining carbon isotope discrimination, Δ, or kinetic isotope effects, α, for CO2-consuming enzymes have traditionally been cumbersome and time-consuming, requiring careful isolation of substrates and products and conversion of these to CO2 for measurement of isotope ratio by mass spectrometry (MS). An equation originally derived by Rayleigh in 1896 has been used more recently to good effect as it only requires measurement of substrate concentrations and isotope ratios. For carboxylation reactions such as those catalysed by d-ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO, EC 4.1.1.39) and PEP carboxylase (PEPC, EC 4.1.1.31), this has still required sampling of reactions at various states of completion and conversion of all inorganic carbon to CO2, as well as determining the amount of substrate consumed. We introduce a new method of membrane inlet MS which can be used to continuously monitor individual CO2 isotope concentrations, rather than isotope ratio. This enables the use of a simplified, new formula for calculating kinetic isotope effects, based on the assumptions underlying the original Rayleigh fractionation equation and given by: --> The combination of inlet membrane MS and this formula yields measurements of discrimination in less than 1 h. We validate our method against previously measured values of discrimination for PEP carboxylase and RuBisCO from several species.

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