The Relationship between Side Reactions and Slow Inhibition of Ribulose-bisphosphate Carboxylase Revealed by a Loop 6 Mutant of the Tobacco Enzyme

Abstract

The first directed mutant of a higher plant ribulosebisphosphate carboxylase/oxygenase (Rubisco), constructed by chloroplast transformation, is catalytically impaired but still able to support the plant's photosynthesis and growth (Whitney, S. M., von Caemmerer, S., Hudson, G. S., and Andrews, T. J. (1999) Plant Physiol. 121, 579 -588). This mutant enzyme has a Leu to Val substitution at residue 335 in the flexible loop 6 of the large subunit, which closes over the substrate during catalysis. Its active site was intact, as judged by its barely impaired competency in the initial enolization step of the reaction sequence, and its ability to bind tightly the intermediate analog, 2-carboxy-D-arabinitol-1,5-bisphosphate. Prompted by observations that the mutant enzyme displayed much less slow inhibition during catalysis in vitro than the wild type, its tendency to catalyze side reactions and its response to the slow inhibitor D-xylulose-1,5-bisphosphate were studied. The lessening in slow inhibition was not caused by reduced production of inhibitory side products. Except for pyruvate production, these reactions were strongly enhanced by the mutation, as was the ability to catalyze the carboxylation of D-xylulose-1,5-bisphosphate. Rather, reduced inhibition was the result of lessened sensitivity to these inhibitors. The slow isomerization phase that characterizes inhibition of the wild-type enzyme by D-xylulose-1,5-bisphosphate was completely eliminated by the mutation, and the mutant was more adept than the wild type in catalyzing the benzylic acid-type rearrangement of D-glycero-2,3-pentodiulose-1,5-bisphosphate (produced by oxidation of the substrate, D-ribulose-1,5-bisphosphate). These observations are consistent with increased flexibility of loop 6 induced by the mutation, and they reveal the underlying mechanisms by which the side reactions cause slow inhibition.Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, 1 EC 4.1.1.39) catalyzes the carboxylation and oxygenation of ribulose-P 2 in photosynthetic CO 2 fixation and photorespiration (1-5). Despite excellent crystal structures of various liganded and unliganded Rubiscos from various sources (6) and intensive mutagenic study of algal (3) and bacterial (7) Rubiscos, progress toward understanding the catalytic mechanism has been hampered by an inability to express the dominant higher plant form of the enzyme in heterologous hosts. This precluded application of the power of directed mutagenesis to the higher plant enzyme until Whitney et al. (8) constructed the first such mutant by chloroplast transformation in the natural host, tobacco. Because it was desirable to maintain photosynthetic viability, a mutation (a Leu to Val substitution at residue 335 of the plastid-encoded large subunit) was chosen with the aim of changing the kinetic parameters (particularly the CO 2 /O 2 specificity (9)) as much as possible without seriously disabling catalytic performance at elevated CO 2 concentration. The substitution successfully reduced the CO 2 /O 2 specificity ...

Differences in Carbon Isotope Discrimination of Three Variants of D-Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Reflect Differences in Their Catalytic Mechanisms

“…The relative energy of the enzyme-bound CKABP intermediate for prokaryotic Rubisco is greater than that for higher plant Rubiscos. In contrast to R. rubrum, L335V mutant tobacco Rubisco appears to bind 2-carboxy-D-arabinitol 1,5-bisphosphate as tightly as WT tobacco Rubisco (61).…”

“…2). In other words, the commitment of the carboxylated intermediate to hydrolysis/cleavage is diminished, even though this intermediate is bound just as tightly by L335V Rubisco as it is by WT tobacco Rubisco (61). Given the relationship between the transition state and the fractionation as argued by Tcherkez et al (29), it is unlikely then that L335V tobacco Rubisco has a lower intrinsic isotope effect associated with the CO 2 addition step (and hence a less product-like transition state for this step).…”

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

“…It should be noted that total 13 C incorporation into organic acids and amino acids will therefore overestimate the flow of 13 C from the CBC. Pyruvate can be formed via PK or from malate via NADP-or NAD-malic enzyme or via a direct side product of Rubisco, in which pyruvate is produced at ;0.7% of the rate of 3PGA (Pearce and Andrews, 2003). It is not facile to use mass spectrometric data to discriminate between the fluxes toward organic acid and amino acid formation via PEPc, malic enzyme, the side reaction of Rubisco, PK, and pyruvate dehydrogenase.…”

Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized

“…This would provide more opportunity for the abortive side reactions emanating from the enediol that progressively inhibit the more CO 2 -specific higherplant Rubisco during catalysis and require the ancillary motor protein, Rubisco activase, to be released (29).…”