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

Abstract

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...

Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

“…␤-Elimination of the 1-phosphate from the initial 2,3-enediol (Fig. 6a) is efficiently suppressed by freezing the geometry of RuPB in a conformation where the bridging oxygen of P1 is in plane with C1, C2, and C3 but can be enhanced in some active site mutants affecting the 1-phosphate binding site (58,59). This reaction yields 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, the same 2,3-diketo intermediate 4 is postulated for DPBS (Fig.…”

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

“…Another pentulose bisphosphate misprotonation product, 3-keto-D-arabinitol-1,5-bisphosphate, has been hypothesized to contribute to fallover inhibition (18,22,24,50). However, its identification as its arabinitol product after dephosphorylation and borohydride reduction is compromised by observations that pentodiulose-P 2 can generate predominantly the same product after similar treatment (14), presumably because of stereochemical bias during reduction.…”

“…The plastome mutant that produces Rubisco with a Leu to Val mutation at residue 335 of the large subunit (8) was grown in an artificially illuminated growth cabinet in an atmosphere containing 0.3% (v/v) CO 2 . Wild-type and L335V Rubiscos were purified either by crystallization as described previously (29) or by polyethylene glycol precipitation and anion-exchange chromatography (Mono Q, Amersham Biosciences) using a procedure similar to that described for spinach Rubisco (18). Tobacco Rubisco concentrations were estimated from the absorbance at 280 nm, assuming that the protein concentration in mg⅐ml Ϫ1 is given by A 280 ϫ 0.7 (30).…”

“…Originating from the Enediol Intermediate-Although the rates of "misfire" reactions originating from the enediol intermediate (Scheme 1 (12,13,18,22,23)) are small compared with the rate of CO 2 addition to this intermediate, they can be measured in the absence of CO 2 and O 2 where consumption of ribulose-P 2 by carboxylation and oxygenation is prevented and the fraction of the enzyme with enediol bound is maximized. Although CO 2 is required to activate Rubisco by carbamylation, this requirement can be fulfilled by preincubation in a HCO 3 Ϫ -containing solution.…”

“…The aci-acid form of P-glycerate (VIII), produced following C2/C3 cleavage of the carboxyketone (VII), ␤ eliminates its phosphate group, forming pyruvate (III). Some of these side reactions are enhanced by certain mutations (12)(13)(14)(15)(16)(17) or when the small subunits are missing (18). In one case, pyruvate production was suppressed by a mutation (12).…”