2′‐Carboxy‐D‐arabitinol 1‐phosphate protects ribulose 1,5‐bisphosphate carboxylase/oxygenase against proteolytic breakdown

Abstract

Trypsin-catalysed cleavage of purified ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the resultant irreversible loss of carboxylase activity were prevented by prior incubation with the naturally occurring nocturnal Rubisco inhibitor 2 H -carboxy-d-arabitinol 1-phosphate (CA1P), as well as with ribulose 1,5-bisphosphate (RuBP), Mg 21 and CO 2 . CA1P also protected Rubisco from loss of activity caused by carboxypeptidase A. When similar experiments were carried out using soluble chloroplast proteases, CA1P was again able to protect Rubisco against proteolytic degradation and the consequent irreversible loss of catalytic activity. Thus, CA1P prevents the proteolytic breakdown of Rubisco by endogenous and exogenous proteases. In this way, CA1P may affect the amounts of Rubisco protein available for photosynthetic CO 2 assimilation. Rubisco turnover (in the presence of RuBP, Mg 21 and CO 2 ) may confer similar protection against proteases in the light. Rubisco is only active when an essential lysine residue within the large subunit is carbamoylated with CO 2 followed by co-ordination of Mg 21 by this carbamate to form a ternary complex at the catalytic site. This process is ordered and reversible, and the equilibrium between the two forms of Rubisco (noncarbamoylated, R 0 , and carbamoylated ternary complex, R) depends on the concentrations of CO 2 and Mg 21 . 2-Carboxy-d-arabitinol 1-phosphate (CA1P) is a naturally occurring transition-state analogue of the carboxylase reaction, which has the capacity to bind tightly to the active site of Rubisco and thus inhibit both the carboxylase and oxygenase activities of the enzyme [3,4]. CA1P is found exclusively in chloroplasts [5] and is synthesized during periods of low light or darkness [6,7]. The amount of CA1P present in leaves is dependent on the plant species and the duration of the dark period [8,9]. In Phaseolus vulgaris, there is enough CA1P present to block all the active sites of Rubisco while in species such as potato, soybean and tobacco there is enough CA1P present to block about 50% of the active sites of Rubisco [6,8]. On transition from dark to light, Rubisco activase promotes the release of CA1P from the catalytic site of Rubisco [10], and free CA1P is rendered noninhibitory by the action of a lightactivated CA1P-phosphatase [11±13]. In vitro, CA1P bound to the active site of Rubisco can be released by treatment with sulfate ions and subsequently removed by gel filtration, to restore the activity of the enzyme [14].During leaf senescence, proteins are catabolized to amino acids, which are transported to the appropriate sinks, which are frequently developing fruits [15,16]. As Rubisco is a major store of nitrogen in actively photosynthesizing mature leaves, the regulation of the breakdown of this enzyme has been subject to considerable study [17±19]. High protease activity has been detected in the vacuole [20], a ubiquitin-targeting system described in the cytosol [21] and light-dependent or ATP-dependent proteolytic breakdown described in ...

The Impacts of Fluctuating Light on Crop Performance

“…The presence of two Rca isoforms with different regulatory properties raises the question of why Rca is regulated at all if constant activation is beneficial for carbon assimilation. The answer may lie, at least in part, in the observation that some inhibitors of Rubisco, like carboxy-D-arabinitol 1-phosphate, bind Rubisco during dark periods and may help protect against proteolysis (Andralojc et al, 1994;Khan et al, 1999). Therefore, given the cost of Rubisco biosynthesis, efforts to improve net assimilation through constant Rubisco activation should examine the impact on Rubisco turnover as well.…”

Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.)

“…The amount of product formed was quantified against an authentic glutamylhydroxamate (Sigma) standard curve. Polypeptides from the crude extract were separated on 10% acrylamide gels and Western blot analyses were performed as described in Habash et al (2001) using a GS specific antibody raised against root nodule GS1 at Rothamsted (Cullimore and Miflin 1984) and a Rubisco antibody raised against wheat rubisco also at Rothamsted (Khan et al 1999). Broad range pre-stained standards (Bio-Rad) were used as markers.…”

CO₂ sequestration in plants: lesson from divergent strategies

“…Some tight binding inhibitors of Rubisco like CA1P (2-carboxyarabinitol-1-phosphate) accumulate under stress conditions and are responsible for low Rubisco activity, release of which can be affected by another enzyme Rubisco activase , Medrano et al 1997. In addition to decreasing the activity of Rubisco such inhibitors also confer protection from oxidative or proteolytic damage (Khan et al 1999). Elucidating biosynthetic and degradative pathways for the synthesis or degradation of these inhibitors can help modulate Rubisco activity and stability (Parry et al 2008).…”