Rubisco side reactions

In the dark, plants also carry out mitochondrial respiration, the oxidation of substrates to C02 and the conversion of 02 to H20. And there is another process in plants that, like mitochondrial respiration, consumes 02 and produces C02 and, like photosynthesis, is driven by light. This process, photorespiration, is a costly side reaction of photosynthesis, a result of the lack of specificity of the enzyme rubisco. In this section we describe this side reaction and the strategies plants use to minimize its metabolic consequences. 

Molecular oxygen usually reacts rapidly with only those organic substrates, such as dihydroflavins, that are able to form stable free radicals. However, the endiolate anion of Eq. 13-50 may be able to donate a single electron to 02 to form a superoxide-organic radical pair prior to formation of the peroxide (see also Eq. 15-30). Similar oxygenase side reactions have been observed for a variety of other enzymes that utilize carbanion mechanisms.283 The reaction of rubisco with 02 is of both theoretical and practical interest, the latter because of its significance in lowering the yield in photosynthesis (Chapter 23). 

The small subunits of rubisco may help suppress undesirable side reactions.285 For example, the following deoxypentodiulose phosphate can be formed by (3 elimination from the second intermediate of Eq. 13-48. 

The key CBB cycle enzyme, RubisCO, is the most abundant protein in the world [8], as it can comprise up to 50% of the total soluble protein in the chloro-plasts or in bacteria using this cycle. This fact is a consequence of the notorious catalytic inefficiency of RubisCO, that is, a low affinity for C02, a slow catalytic turnover rate, and a wasteful oxygenase side reaction responsible for photorespiration, resulting in a futile cleavage of the substrate to form phosphoglycolate as a side product. However, the CBB cycle enzymes are oxygen-insensitive and can easily be controlled, because the whole pathway is separated from... 

Completion of a catalytic cycle by Rubisco requires stabilization of several inherently unstable intermediates. Imperfect stabilization of these intermediates is reflected in non-productive side reactions, including several involving the enediol(ate) (I). Not only does the formation of side products provide insight into limitations of Rubisco efficiency in vivo, but perturbation of Aese side reactions by mutants can reveal amino acid groups cmcial to intermediate stabilization. 

The most prominent side reaction of Rubisco is its counterproductive oxygenase activity, reflecting competition with CO2 for the enediol(ate) intermediate (1) (23). Partitioning between the two pathways (vjvo) is defined by Vc/Vo = T ([C02]/[02]), where x is VJCJVoK,. (24). Because x can be interpreted in terms of the free energy differential for carboxylated versus oxygenated transition states (25-26), it provides insight into determinants of Rubisco specificity. 

Another potential side reaction of the enediol(ate) intermediate is formation of the dicarbonyl compound, l-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, resulting from p-elimination of the Cl-phosphate due to improper stabilization and/or premature dissociation of enediol(ate) from the enzyme active site. This compound has been characterized by reduction with borohydride, oxidation with H2O2, complexation with o-phenylenediamine, and 13C-NMR (23, 34). The p-elimination product is not detected in reactions with wild-type R. rubrum Rubisco but is formed in substantial amounts with mutants in which the Cl-phosphate ligands are substituted, demonstrating the required role of these amino acid side chains in stabilizing the enediol(ate) intermediate (34-35). 

Figure 20.6. A Wasteful Side Reaction. The reactive enediolate intermediate on rubisco also reacts with molecular oxygen to form a hydroperoxide intermediate, which then proceeds to form one molecule of 3-phosphogly cerate and one molecule of phosphoglycolate.

Central to the proposed mechanism for plant rubisco is a carbamoylated Lys side chain with a bound Mg2+ ion. The Mg2+ ion brings together and orients the reactants at the active site (Fig. 20-6) and polarizes the C02, opening it to nucleophilic attack by the five-carbon enediolate reaction intermediate formed on the enzyme (Fig. 20-7). The resulting six-carbon intermediate breaks down to yield two molecules of 3-phosphoglycerate. 

The carbanion of the remaining three-carbon fragment is proto-nated by the nearby side chain of Lys175, generating a second molecule of 3-phosphoglycerate. The overall reaction therefore accomplishes the combination of one C02 and one ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate, one of which contains the carbon atom from C02 (red). 0 Rubisco Mechanism Rubisco Tutorial... 

In this chapter, we present three approaches to address mechanistic issues with Rubisco mutants characterization of catalysis of partial reactions, analysis of side products, and subtle alteration of the active-site microenvironment by manipulation with exogenous reagents. 

This salvage pathway serves to recycle three of the four carbon atoms of two molecules of glycolate. However, one carbon atom is lost as CO2. This process is called photorespiration because O2 is consumed and CO2 is released. Photorespiration is wasteful because organic carbon is converted into CO2 without the production of ATP, NADPH, or another energy-rich metabolite. Evolutionary processes have presumably enhanced the preference of rubisco for carboxylation. For instance, the rubisco of higher plants is eightfold as specific for carboxylation as that of photosynthetic bacteria. However, some oxygenase activity may be an inevitable side effect of the carboxylase reaction mechanism. 

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