Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.The universal CO 2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (EC 4.1.1.39) catalyzes the primary reactions of photosynthesis and photorespiration by carboxylation and oxygenation of ribulose-1,5-bisphosphate (RuBP), respectively. The RuBisCO from cyanobacteria, algae (excluding some dinoflagellates), and higher plants is a hexadecamer consisting of eight large and eight small subunits organized as four protomers, each of which has two active sites at the interface between each large subunit pair (3,4,29). In the RuBisCO of many species, an intradimeric disulfide bond is formed under oxidizing conditions between the Cys247 residues of a large subunit pair (30). A sequence comparison of more than 500 RuBisCO genes revealed high levels of similarity, particularly among large subunits, and residues that form the catalytic site are entirely conserved (21). As a result of the enzymatic inefficiency manifested by a low catalytic turnover number (1.5 to 12 carboxylations s Ϫ1 catalytic site Ϫ1 [5,24]), as well as a low affinity for substrate CO 2 and competition between the CO 2 and O 2 substrates, it is accepted that the activity of RuBisCO limits both photosynthesis and photorespiration under various ecophysiological conditions (40).Due to the pivotal role of RuB...

Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+channels as a novel pharmacological target and provides a rational framework for drug design.

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...

Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (Kcat) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40–50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced Kcat so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (Pmax) under saturating irradiance and Ci concentrations indicate that a >60% reduction in Kcat can be tolerated before Pmax in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH.

No abstract