Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis

Paul, Kalanethee; Mk, Morell; Tj, Andrews

doi:10.1021/bi00105a029

Cited by 35 publications

(37 citation statements)

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“…Carboxylase Assay-The carboxylase activity of L 8 Rubisco was routinely measured by 14 CO 2 fixation both in the presence and in the absence of added small subunits (19,29 ). After preincubation for 10 min at 25°C, 4 units⅐ml Ϫ1 of rabbit muscle lactate dehydrogenase (Boehringer Mannheim) and 0.2 mM NADH were added, and catalysis was initiated by adding 2 mM ribulose-P 2 .…”

Section: Methodsmentioning

confidence: 99%

Side Reactions Catalyzed by Ribulose-bisphosphate Carboxylase in the Presence and Absence of Small Subunits

Morell¹,

Wilkin²,

Kane

et al. 1997

Journal of Biological Chemistry

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The large subunit core of ribulose-bisphosphate carboxylase from Synechococcus PCC 6301 expressed in Escherichia coli in the absence of its small subunits retains a trace of carboxylase activity (about 1% of the k cat of the holoenzyme) ( the addition of CO 2 to ribulose-P 2 , producing two molecules of P-glycerate using a multistep reaction sequence involving at least three enzyme-bound catalytic intermediates (Scheme 1) (for reviews, see Refs. 1-3). Two of these intermediates, the first and the third, are strong nucleophiles by virtue of their enediol character. Both are susceptible to side reactions that abort the carboxylation sequence and compromise the catalytic efficiency of the enzyme. The first of these intermediates is the 2,3-enediol form of ribulose-P 2 produced by removal, by an enzymatic base, of the proton attached to C-3 of ribulose-P 2 . There may be two or more differently protonated forms of this intermediate; only the enediol form is shown in Scheme 1. Collectively, we refer to all forms of this intermediate as "the enediol." This is the species that must be attacked by CO 2 at C-2 and (concertedly or sequentially) by H 2 O at C-3 to form the six-carbon intermediate and thus allow carboxylation to proceed (Scheme 1). Three different classes of side reactions are known for this intermediate. First, the enediol is also attacked by O 2 at C-2, resulting in the formation of 2-phosphoglycolate which is, in turn, partially recycled to photosynthetic metabolism by the photorespiratory glycolate pathway (4 -6). Second, the enediol can be reprotonated incorrectly, producing pentulose bisphosphate isomers of the substrate (xylulose-P 2 and 3-ketoarabinitol-P 2 ) (7-12) which are strong inhibitors and/or very weak substrates of the enzyme (13-15). Third, mutants of Synechococcus PCC 6301 and Rhodospirillum rubrum Rubiscos were discovered that were compromised in their ability to stabilize the enediol. These mutant enzymes catalyzed the production of varying amounts of deoxypentodiulose-P as a result of ␤ elimination of the phosphoryl group attached to C-1 of the intermediate (P1) (16,17).The final intermediate in the carboxylation sequence is produced following cleavage of the C-2/C-3 bond of the six-carbon intermediate (Scheme 1). This aci-acid form of the P-glycerate molecule, derived from carbons 1 and 2 of ribulose-P 2 and the incoming CO 2 molecule, requires stereospecific protonation at C-2 to form P-glycerate. Only a single kind of side reaction is known for this intermediate, ␤ elimination of the phosphoryl moiety to produce enol-pyruvate and thus pyruvate (18). All wild-type Rubiscos studied so far partition approximately 0.7% of their aci-acid intermediate to pyruvate (18), and mutants are known in which this partitioning ratio is increased or decreased (14, 16, 17). Thus both of Rubisco's enediol-like intermediates are prone to ␤ elimination of the P1 phosphate group, * This work was supported by Australian National University's Center for Molecular Structure and Function. The costs of public...

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Section: Methodsmentioning

confidence: 99%

Side Reactions Catalyzed by Ribulose-bisphosphate Carboxylase in the Presence and Absence of Small Subunits

Morell¹,

Wilkin²,

Kane

et al. 1997

Journal of Biological Chemistry

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“…Peak fractions were frozen in liquid N 2 and stored at Ϫ80°C. The Michaelis constants (at pH 8.3) for CO 2 , O 2 , and ribulose-P 2 (Paul et al, 1991) and the CO 2 /O 2 specificity (Kane et al, 1994) were measured using these purified preparations.…”

Section: Purification and Assay Of Rubiscomentioning

confidence: 99%

Directed Mutation of the Rubisco Large Subunit of Tobacco Influences Photorespiration and Growth

et al. 1999

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The gene for the large subunit of Rubisco was specifically mutated by transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO 2 enrichment. In 0.3% (v/v) CO 2 , the mutant and wildtype plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO 2 -fixing rate and its ability to distinguish between CO 2 and O 2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO 2 , O 2 , and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O 2 concentrations. Consistent with the reduced CO 2 fixation rate at saturating CO 2 , the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed.

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“…Rubisco from Rhodospirillum rubrum, which is the prokaryotic enzyme used most extensively for directed mutagenesis studies (4), lacks small subunits. Directed mutagenesis of cyanobacterial small subunits, expressed and assembled with cyanobacterial large subunits in E. coli, has shown that small subunits can influence holoenzyme stability and the rate of carboxylation (15)(16)(17)(18). However, such studies cannot assess the significance of the structural and functional differences between prokaryotic and eukaryotic Rubisco holoenzymes.…”

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confidence: 99%

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

Khrebtukova

Spreitzer

1996

Proc. Natl. Acad. Sci. U.S.A.

108

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Ribulose-1,5-bisphosphate carboxylase͞ oxygenase (EC 4.1.1.39) is the key photosynthetic enzyme that catalyzes the first step of CO 2 fixation. The chloroplastlocalized holoenzyme of plants and green algae contains eight nuclear-encoded small subunits and eight chloroplastencoded large subunits. Although much has been learned about the enzyme active site that resides within each large subunit, it has been difficult to assess the role of eukaryotic small subunits in holoenzyme function and expression. Small subunits are coded by a family of genes, precluding genetic screening or nuclear transformation approaches for the recovery of small-subunit mutants. In this study, the two small-subunit genes of the green alga Chlamydomonas reinhardtii were eliminated during random insertional mutagenesis. The photosynthesis-deficient deletion mutant can be complemented with either of the two wild-type small-subunit genes or with a chimeric gene that contains features of both. Thus, either small subunit is sufficient for holoenzyme assembly and function. In the absence of small subunits, expression of chloroplast-encoded large subunits appears to be inhibited at the level of translation.

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Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis

Cited by 35 publications

References 48 publications

Side Reactions Catalyzed by Ribulose-bisphosphate Carboxylase in the Presence and Absence of Small Subunits

Side Reactions Catalyzed by Ribulose-bisphosphate Carboxylase in the Presence and Absence of Small Subunits

Directed Mutation of the Rubisco Large Subunit of Tobacco Influences Photorespiration and Growth

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

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