Stimulation of Ribulose Bisphosphate Carboxylase Activity by Inorganic Orthophosphate without an Increase in Bound Activating CO<sup>2</sup>: Co-operativity between the Subunits of the Enzyme

Parry, M. A. J.; Schmidt, C. N. G.; Cornelius, M. J.; Keys, A. J.; Millard, B. N.

doi:10.1093/jxb/36.9.1396

Cited by 38 publications

(30 citation statements)

References 18 publications

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“…The elevated level of RuBP observed after 18 min at 21 mbar 02 (Table I) or in chloroplasts in limiting Pi concentration (1 1) indicates that Rubisco has decreased in its activity in both cases. This is in agreement with earlier findings that Pi is required for maximal activation of Rubisco (2,11,15).…”

Section: = (2)supporting

confidence: 94%

Limitation of Photosynthesis by Carbon Metabolism

Sharkey¹,

Stitt²,

Heineke³

et al. 1986

Plant Physiol.

232

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The occurrence of O2-insensitive photosynthesis at high quantum flux and moderate temperature in Spinacia oleracea was characterized by analytical gas exchange measurements on intact leaves. In addition photosynthetic metabolite pools were measured in leaves which had been rapidly frozen under defined gas conditions. Upon switching to low 0°in 02-insensitive conditions the ATP/ADP ratio fell dramatically within one minute. The P-glycerate pool increased over the same time. Ribulose bisphosphate initially declined, then increased and exceeded the pool size measured in air. The pools of hexose monophosphates and UDPglucose were higher at a partial pressure of 02 of 21 millibars than at 210 millibars. These results are consistent with the hypothesis that the rate of sucrose synthesis limited the overall rate of assimilation under O2-insensitive conditions.In this article we ask whether manipulating the ambient 02 concentration provides a way of recognizing a situation in which sucrose or starch synthesis limit the rate of photosynthesis. During photosynthesis, CO2 and Pi are converted to triose-P in the chloroplast. The triose-P is converted to sucrose in the cytosol, or starch in the chloroplast, liberating Pi for use in subsequent CO2 fixation. The production of triose-P in the chloroplast requires electron transport, regeneration of RuBP3 and activity of Rubisco. As all these processes are also needed for photorespiration, there is normally a stimulation of photosynthesis when the ambient 02 concentration is lowered to suppress photorespiration. However, photorespiration does not compete for the reactions by which triose-P is converted into end products such as sucrose and starch. This means that the rate of photosynthesis would not be reduced by photorespiration when photosynthesis is limited by the utilization of photosynthesis products provided there is adequate electron transport and carbon reduction cycle activity to cope with the additional fluxes required when photorespiration occurs. The rate of photosynthesis would then be independent of the ambient 02 level. It has been observed that decreasing the 02 concentration from 210 to 21 mbar does not always lead to an increase of photosynthesis, especially when the light intensity is high and the temperature is below 20°C (3,12,14,24). It has been proposed that this 02 insensitivity of photosynthesis is due to a limitation of photosynthate utilization, in particular triose-P utilization for sucrose and starch synthesis (17, 18). When 02 sensitivity is lost photosynthesis also fails to respond to CO2 (18) indicating that the phenomenon is not simply an effect of 02 on pseudo-cyclic photophosphorylation.A limitation ofthe utilization of phosphorylated intermediates would lead to a fall of the stromal Pi. Many studies have shown that the rate of CO2 fixation in isolated chloroplasts depends on the stromal Pi level. Upon lowerng the stromal Pi concentration from 7 to 2 mM, CO2 fixation was decreased by 40% (11) and this inhibition of CO2 fixation was acco...

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Section: = (2)supporting

confidence: 94%

Limitation of Photosynthesis by Carbon Metabolism

Sharkey¹,

Stitt²,

Heineke³

et al. 1986

Plant Physiol.

232

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“…3A and B), a phenomenon previously reported by Anwaruzzaman et al (2) and Marcus and Gurevitz (16). Under these conditions P i has two effects on Rubisco activation: it raises the carbamylation level, but also elevates the activity of the carbamylated enzyme (16,21). Whereas neutralization of a single positive charge at the latch site (substitution K305A or H310A) had no effect, the double mutation K305A/H310A had a clear inhibitory effect, especially at low P i concentrations (Fig.…”

Section: Vol 187 2005 Rubisco Regulation By Orthophosphatementioning

confidence: 94%

“…It is accepted that these compounds evoke activation by elevating the carbamylation level, presumably by slowing the rate of enzyme deactivation (3, 12, 18). Nonetheless, evidence was brought that in addition to stimulation of Rubisco carbamylation, P i also enhances the activation of the enzyme without a parallel increase in the carbamylation level (16,21). Whereas McCurry et al (18) reported monophasic, hyperbolic P i concentration-dependent activation of Rubisco, nonmonotonously biphasic kinetics was obtained in other, more recent studies (2, 16), indicating that P i stimulates enzyme activation via a mechanism that involves multiple interacting sites (28).…”

mentioning

confidence: 97%

“…Several effectors, mostly anions such as orthophosphate (P i ), sugar-phosphates, sulfate, and NADPH, modulate the activation process via a mechanism that is still controversial (2,3,5,11,12,16,18,20,21,26). It is accepted that these compounds evoke activation by elevating the carbamylation level, presumably by slowing the rate of enzyme deactivation (3,12,18).…”

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confidence: 99%

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Mutagenesis at Two Distinct Phosphate-Binding Sites Unravels Their Differential Roles in Regulation of Rubisco Activation and Catalysis

et al. 2005

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Orthophosphate (P i ) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P i at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P i -stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P i inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K m (RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P i binding only at the 5P site inhibits catalysis in a competitive manner.Activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC4.1.1.39) is essential for catalysis and occurs by carbamylation of Lys-201 and binding of Mg 2ϩ by the carbanion (14). Several effectors, mostly anions such as orthophosphate (P i ), sugar-phosphates, sulfate, and NADPH, modulate the activation process via a mechanism that is still controversial (2,3,5,11,12,16,18,20,21,26). It is accepted that these compounds evoke activation by elevating the carbamylation level, presumably by slowing the rate of enzyme deactivation (3, 12, 18). Nonetheless, evidence was brought that in addition to stimulation of Rubisco carbamylation, P i also enhances the activation of the enzyme without a parallel increase in the carbamylation level (16,21). Whereas McCurry et al. (18) reported monophasic, hyperbolic P i concentration-dependent activation of Rubisco, nonmonotonously biphasic kinetics was obtained in other, more recent studies (2, 16), indicating that P i stimulates enzyme activation via a mechanism that involves multiple interacting sites (28).Paradoxically, in addition to the stimulatory effect on activation, the effectors also inhibit the catalytic activity of the enzyme by competing with the substrate ribulose 1,5-bisphosphate (RuBP) (3, 16). Based on the observation of McCurry et al. (18) and the ability of the transition state analog carboxyarabinitol-bisphosphate to prevent 6-phosphogluconate from binding, Badger and Lorimer (3) suggested that inhibition of activity and stimulation of activation are both induced by effector binding to the catalytic site. Although this would imply that RuBP competitively inhibits the stimulation...

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“…For (8,18). If '4CO2 is used in the assay, the label which remains with the high mol wt fraction following carboxyarabinitol-P2 trapping and gel filtration gives a measure of the proportion of carbamylated, Mg24-bound enzyme (9,14,17). Since carboxyarabinitol-P2 binds more tightly to ECM than to E, it promotes carbamylation.…”

mentioning

confidence: 99%

Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Not Due to Decarbamylation of the Catalytic Site

Edmondson¹,

Badger²,

Andrews³

1990

Plant Physiol.

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An investigation was made of the proposal that the slow inactivation of ribulosebisphosphate carboxylase (Rubisco) activity, which occurs during in vitro assays, is due to decarbamylation of the enzyme. The level of carbamylation was compared with catalytic activity during assay conditions in which activity was both increasing and decreasing. Carbamylation level was measured using the reaction-intermediate analogue 2'-carboxy-D-arabinitol-1,5-bisphosphate (carboxyarabinitol-P2). A dual isotope procedure was used in which [3H]carboxyarabinitol-P2 measured total active sites and 14CO2 reported the level of carbamylation.The efficacy of the procedure was verified both in the presence and in the absence of the substrate D-ribulose-1,5-bisphosphate (ribulose-P2). These measurements showed that changes in activity during assays were not correlated with carbamylation status. Inactivation during assays initiated with both fully and partially carbamylated enzyme was not associated with any change in carbamylation level. This implies that the loss of activity during assays is not due to ribulose-P2 binding and sequestering the E form of the enzyme. Ribulose-P2 did not appear to alter the equilibrium between carbamylated and uncarbamylated enzyme, but it did slow the rate at which enzyme was both decarbamylated and carbamylated. The most likely explanation for the loss of activity during assays appears to be the sequestration of carbamylated, Mg2 -bound active sites by an inhibitor. (ECM). This theory has been based on experimental findings which show that phosphorylated sugars, including ribulose-P2, may bind to both the E and ECM forms of the enzyme at the catalytic site (2, 14). When bound to the ECM form, the activating CO2 and Mg2' are stabilized on the enzyme. When bound to the E form, carbamylation is retarded. Depending on the relative affinity ofthese compounds for E and ECM, they may shift the equilibrium between these forms of the enzyme.There have been several indications that ribulose-P2 binds tightly to the E form of Rubisco. For example, Jordan and Chollet (9) measured a dissociation constant for the E-ribulose-P2 complex of 21 nM at 2°C, and Laing and Christeller ( 11) showed that activation of E was very slow in the presence of ribulose-P2. With such tight binding of ribulose-P2 to E, it could readily be expected that there should be a decline in the level of ECM during carboxylation, and conversely, that there should be very little increase in the level of ECM when E is added to ribulose-P2 in the presence of CO2 and Mg2'. In fact, it is thought that the enzyme, Rubisco activase, is required in vivo to promote activation in the presence of ribulose-P2 (20). Contrary to this, there are a number of reports of ribulose-P2 promoting carbamylation of Rubisco. Vater et al. (22) showed that ribulose-P2 induced a decrease in the dissociation constant for the EC complex similar to that seen with sugar phosphate activators such as 6-phosphogluconate, and NADPH (2,5,14). Additionally, Laing and Christelle...

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Stimulation of Ribulose Bisphosphate Carboxylase Activity by Inorganic Orthophosphate without an Increase in Bound Activating CO²: Co-operativity between the Subunits of the Enzyme

Cited by 38 publications

References 18 publications

Limitation of Photosynthesis by Carbon Metabolism

Limitation of Photosynthesis by Carbon Metabolism

Mutagenesis at Two Distinct Phosphate-Binding Sites Unravels Their Differential Roles in Regulation of Rubisco Activation and Catalysis

Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Not Due to Decarbamylation of the Catalytic Site

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Stimulation of Ribulose Bisphosphate Carboxylase Activity by Inorganic Orthophosphate without an Increase in Bound Activating CO2: Co-operativity between the Subunits of the Enzyme

Cited by 38 publications

References 18 publications

Limitation of Photosynthesis by Carbon Metabolism

Limitation of Photosynthesis by Carbon Metabolism

Mutagenesis at Two Distinct Phosphate-Binding Sites Unravels Their Differential Roles in Regulation of Rubisco Activation and Catalysis

Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Not Due to Decarbamylation of the Catalytic Site

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Stimulation of Ribulose Bisphosphate Carboxylase Activity by Inorganic Orthophosphate without an Increase in Bound Activating CO²: Co-operativity between the Subunits of the Enzyme