A Kinetic Characterization of Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis

Edmondson, Daryl L.; Badger, Murray R.; Andrews, T. John

doi:10.1104/pp.93.4.1376

Cited by 69 publications

(76 citation statements)

References 25 publications

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“…The ratio of the activity in the linear phase to that in the burst increased from 0.4 to 0.75 with increasing concentrations of 6-P-Gln. A similar increase of the activity in the linear phase in the presence of 6-P-Gln had been observed by Edmondson et al [19]. The plot of the ratio against the 6-PGln concentration was sigmoidal.…”

Section: Resultssupporting

confidence: 67%

Regulation of the activity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase through cooperative binding of 6‐phosphogluconate to its regulatory sites

Yokota

Higashioka

Wadano

1992

European Journal of Biochemistry

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This study was attempted to elucidate the mechanism of the regulation of the turnover number on the catalytic sites by the regulatory sites of spinach ribulose-l,5-bisphosphate carboxylase/oxygenase [Rbu(l,5)P2CO]. To this end, we analyzed the effects of the binding of 6-phosphogluconate (6-PGln) to the regulatory sites of the enzyme on the progress in the subsequent catalysis assayed with 0.5 mM ribulose 1,5-bisphosphate [Rbu(l,5)P2]. This concentration of Rbu(1,5)P2 hardly binds to the regulatory sites but competes with 6-P-Gln for the catalytic sites. An equilibrium binding assay showed that Rbu(l,5)P2C0 bound 8 mol6-P-Gln/mol enzyme in addition to the catalytic sites. The binding to the eight regulatory sites was positively cooperative. The biphasic reaction course, inherent in the carboxylase reaction of plant Rbu(l,5)P2C0 and composed of a burst for an initial few minutes and a subsequent linear phase, became faint with increasing binding of 6-P-Gln to the sites. The change of the reaction progress from the biphasic to linear course was ascribed to the suppression of the functioning form of the enzyme from the high-activity to low-activity form and to the increased turnover number on the catalytic sites though the binding of 6-P-Gln to the regulatory sites. Ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu-(1 ,5)P2CO] from most photosynthetic organisms is composed of eight each of large and small subunits [l]. The large subunits participate in the catalysis of C 0 2 and O2 fixation in photosynthesis and photorespiration, respectively [2]. Recent crystallographic analyses of Rbu(1 ,5)P2co have emphasized the direct linkage between neighboring large subunits and the indirect linkage through the small subunits [3]. It has now become a realistic possibility that metabolically important functions on the large subunits may have a cooperative nature. However, this nature had remained unclear in catalysis until the recent discovery of hysteresis of Rbu(l,5)P2C0.The carboxylase reaction of Rbu(1 ,5)P2co from plant sources slows down to a steady level with reaction time [2,[4][5][6][7][8][9][10][11][12]. The decrease during the reaction results from the hysteretic properties of Rbu(l,5)PzC0 (E) [lo -121. The form of the carboxylase activated with COz (C) and Mg2+ (M), ECM, binds Rbu(1 ,5)P2 on the substrate-binding catalytic sites in the pesence of less than 1 mM Rbu(l,5)P2. The resulting form, ECMSR [SR is Rbu(1,5)P2 bound to the catalytic sites], is obliged to go through a slow conformational change to

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Section: Resultssupporting

confidence: 67%

Regulation of the activity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase through cooperative binding of 6‐phosphogluconate to its regulatory sites

Yokota

Higashioka

Wadano

1992

European Journal of Biochemistry

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“…This inactivation, or "fallover," continues until eventually a steady rate of catalysis is reached which is substantially less than the initial rate (6). The phenomenon is not a result of substrate depletion or product accumulation and it is worsened by lowering the pH or the CO2 concentration and alleviated by inhibitors which bind at the active site (6).…”

Section: Methodsmentioning

confidence: 99%

Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Caused by Accumulation of a Slow, Tight-Binding Inhibitor at the Catalytic Site

Edmondson¹,

Badger²,

Andrews³

1990

Plant Physiol.

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The slow inactivation which accompanies catalysis by higherplant ribulose-P2 carboxylase-oxygenase (Rubisco) (2), such reactions could occur at the catalytic site as well as in solution.In this study, we show that a slow, tight-binding inhibitor is, indeed, responsible for fallover. The data are most consistent with the inhibitor being formed as a by-product during catalysis. MATERIALS AND METHODSRubisco2 becomes slowly inactivated during catalysis after exposure to ribulose-P2. This inactivation, or "fallover," continues until eventually a steady rate of catalysis is reached which is substantially less than the initial rate (6). The phenomenon is not a result of substrate depletion or product accumulation and it is worsened by lowering the pH or the CO2 concentration and alleviated by inhibitors which bind at the active site (6). Earlier papers of this series indicated that it is unlikely that fallover is caused by readily reversible, inhibitory binding of ribulose-P2 at a non-catalytic, regulatory site (6) and showed that fallover is not due to decarbamylation of the active-site E-carbamyl-lysyl residue that is required for catalysis (7).One possibility which remains is that fallover is caused by ' Present address:

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“…For the spinach enzyme, this might be a result of inhibition caused by accumulating misprotonation by-products. Indeed, the kinetics of the decline for the spinach enzyme appeared quite reminiscent of the decline usually seen during catalysis which has been attributed to that cause (7)(8)(9)(10). Synechococcus Rubisco, however, is not subject to progressive inactivation during catalysis, at least at CO 2 saturation (16), and it showed a more pronounced decline such that, after 15-20 min, the rate of absorbance increase had fallen to approach the basal rate seen in the enzyme-free control.…”

Section: Conditions Leading To Exhaustion Of Gaseous Substrates-un-mentioning

confidence: 99%

“…Rubisco holoenzyme (L 8 S 8 ) expressed in E. coli from the rbcL and rbcS genes of Synechococcus PCC 6301 was purified as described (16). Spinach Rubisco was purified using a procedure involving polyethylene glycol precipitation followed by anion-exchange chromatography on a Waters Protein-Pak Q column, essentially as described by Edmondson et al (7) but omitting the final gel filtration step.…”

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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A Kinetic Characterization of Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis

Cited by 69 publications

References 25 publications

Regulation of the activity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase through cooperative binding of 6‐phosphogluconate to its regulatory sites

Regulation of the activity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase through cooperative binding of 6‐phosphogluconate to its regulatory sites

Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Caused by Accumulation of a Slow, Tight-Binding Inhibitor at the Catalytic Site

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

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