Bioinformatic tools uncover the C-terminal strand of Rubisco's large subunit as hot-spot for specificity-enhancing mutations.

Rubisco assumes the double role of accumulating biomass by fixing carbon dioxide to ribulose-1,5-bisphosphate and binding of molecular oxygen to the same substrate. The specificity factor of this mutually competitive activity, defined as the ratio of carboxylation to oxygenation efficiency, varies considerably for reasons which remain obscure. The explanation and the enhancement of specificity are of high theoretical and practical interest. Despite a wealth of structures and experimental findings, the systematic analysis of available data is still at its beginning. Here, we (a) present an analysis of sequences of the large subunit which reliably finds specificity-enhancing mutations and ranks them according to the probability of success. For mutations near the C-terminus, we (b) show by simulations that the positive influence they have on specificity can be explained by the time-window hypothesis.

Monitoring cytosolic pH of carboxysome-deficient cells of Synechocystis sp. PCC 6803 using fluorescence analysis.

Disruption of the ccmM gene in the cyanobacterium Synechocystis sp. PCC 6803 causes a deficiency of carboxysomes and impairs growth in ambient CO2. The effect of this gene defect on cellular metabolism was investigated using electron microscopy, biochemical and fluorescence analysis. Mutant cells were devoid of the characteristic dense polyhedral bodies called carboxysomes. The photosynthetic oxygen evolution was considerably lower in mutant cells compared to wild type, while Rubisco activity in cell extracts was similar. During photosynthetic CO2-dependent oxygen evolution, Rubisco Vmax dropped from 142 micromol mg-1 chlorophyll h-1 (WT) to 77 micromol mg-1 chlorophyll h-1 in the mutant cells, and the Km for Ci (inorganic carbon) increased from 0.5 mM (WT) to 40 mM. The fluorescent indicator, acridine yellow, was used for non-invasive measurements of cytoplasmic pH changes in whole cells induced by addition of Ci, making use of the decrease in fluorescence yield that accompanies cytoplasmic acidification. The experimental results indicate that control of the cytoplasmic pH is linked to the internal carbon pool (Ci). Both wild-type and ccmM-deficient cells showed a linear response of acridine yellow fluorescence quenching and, thus, of internal acidification, with respect to externally added inorganic carbon. However, the fluorescence analysis of mutant (carboxysome-free) cells indicated slower kinetics of Ci accumulation.

The kinetics of conformation change as determinant of Rubisco's specificity.

The molecular basis of Rubisco's specificity is investigated in terms of the structure and kinetics of the enzyme. We propose that the rates of the conformational changes (closing/opening) of the binding niche exert a crucial influence on apparent binding rates and the enzyme's specificity. An extended reaction scheme for binding and conformational kinetics is presented and expressed in a mathematical model. The closed conformation, known from X-ray structures, is assumed to be necessary for binding of the gaseous substrates (carbon dioxide and oxygen) and for catalysis. Opening the niche interrupts catalysis and enables a fast exchange of those molecules between the internal cavity and the surrounding solvent. Our model predicts that specificity of Rubisco for CO(2) increases with the rate by which the niche opens. This is due to the fact that binding of the carbon dioxide is faster than oxygen binding, which is hampered by spin inversion. The apparent rate of carbon dioxide binding correlates with the repetition rate of the conformational change, and the rate of oxygen binding with the probability of the closed state.

Simple Determination of the CO(2)/O(2) Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [C]Glycerate 3-Phosphate.

A new method is presented for measurement of the CO(2)/O(2) specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [(14)C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. (14)CO(2) fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO(2) in O(2)-saturated water and carboxylase only with 160 micromolar CO(2) under N(2). Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [(14)C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 +/- 4), from the green alga Chlamydomonas reinhardtii (66 +/- 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.

Aspartate aminotransferases ofPanicum miliaceum L. andPanicum antidotale retz. : Inactivation and reconstitution.

L-Aspartate: 2-oxoglutarate transaminase was isolated and partially purified from leaves ofPanicum miliaceum (C4, NAD-malic enzyme type) and ofPanicum antidotale (C4, NADP-malic enzyme type). In each preparation two isoenzymes with different kinetic properties could be characterized. The enzyme activity was irreversibly inhibited by 2-aminooxyacetic acid and by 2-amino-4-methoxy-3-butenoic acid. The first inhibitor reacted with pyridoxal 5-phosphate, and its inhibition could be reversed by the exchange of the modified coenzyme. The second inhibitor binds not only to the coenzyme pyridoxal 5-phosphate, but also to the apoprotein. The results of the dissociation and reconstitution experiments were in agreement with the kinetic data, showing that the mode of inactivation was different for 2-aminooxyacetic acid and 2-amino-4-methoxy-3-butenoic acid.

The oxygen-dependent deactivation and reactivation of spinach ribulose-1,5-bisphosphate carboxylase.

Ribulose-1,5-bisphosphate carboxylase-oxygenase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) is deactivated by the removal of oxygen, and reversibly reactivated by its readdition to the enzyme solution. A short pulse of oxygen to the anaerobic enzyme solution is sufficient to trigger the reactivation process; the Ka value for this reaction was estimated as 0.12 mM oxygen. The enzyme could not be reactivated under anaerobic conditions by an organic oxidant (benzoylperoxide) or by sulfhydryl group reducing reagents (dithiothreitol or beta-mercaptoethanol), suggesting that the process of reactivation was oxygen specific. Furthermore, the inhibition of the reactivation by superoxide anion scavengers such as Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid), copper penicillamine, hydroxylamine, nitroblue tetrazolium, and ascorbate, indicated that the monovalent reduced oxygen was involved as the reacting species in this process. The deactivation of the enzyme associated with the removal of oxygen was also sensitive to the presence of scavengers of O2(-), suggesting that superoxide anion was also involved in the deactivation process. Both the carboxylase and the oxygenase activities were similarly affected under all the experimental conditions studied. On the basis of these results it is argued that the enzyme molecules are able to reduce oxygen and that superoxide anion causes the deactivation or reactivation of the enzyme.

The activation of ribulose-1,5-bisphosphate carboxylase-oxygenase from spinach by oxygen.

The effect of oxygen on ribulose-1,5-bisphosphate carboxylase-oxygenase from spinach was investigated. Both activities were deactivated by removal of oxygen and reversibly reactivated by oxygenation of the enzyme solution. The change in enzyme activities was accompanied by conformational changes as studied by the use of intrinsic and extrinsic fluorescent probes. The analysis of cysteine sulfhydryl groups accessible to 5,5'-dithiobis-(2-nitrobenzoic acid) revealed that the number of these groups changed with the oxygen concentration. The kinetic of the exposure of eight cysteine residues was similar to the loss of enzyme activities. The modification of these groups with 5,5'-dithiobis-(2-nitrobenzoic acid) caused almost complete loss of both the activities. The enzyme isolated from a photolithotrophic organism, Chromatium vinosum, was not affected by oxygen removal. During air--argon transitions, neither the enzyme conformation nor the number of accessible sulfhydryl groups changed.

Effects of glycidate on carbon dioxide fixation with isolated spinach chloroplasts.

Glycidate (2,3-epoxypropionate) stimulated CO(2) fixation in isolated spinach chloroplasts up to 100%. In the presence of glycidate the initial lag phase was abolished and the chloroplasts exported mainly 3-phosphoglycerate instead of dihydroxyacetone phosphate.Glycolate formation was not inhibited by glycidate, which is in agreement with the observation that preactivated ribulose-1,5-bisphosphate oxygenase is not inhibited by this compound. Furthermore, glycidate had no effect on electron transport (NADP reduction) and photophosphorylation, nor was a change in the coupling ratio observed.

The effect of divalent metal ions on the activity of Mg(++) depleted ribulose-1,5-bisphosphate oxygenase.

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg(++). The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO2. Incubation with Mg(++) and CO2 restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn(++), Co(++), and Ni(++)) and CO2 fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO2-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn(++) ions were present. Other divalent cations such as Ca(++) and Zn(++), restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn(++) to the Mg(++) and CO2 preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.

The use of 1-anilino-8-naphthalene sulfonate as fluorescent probe for conformational studies on ribulose-1,5-bisphosphate carboxylase.

The influence of Mg2+ ions and temperature on the structure of the enzyme ribulose-1,5-bisphosphate carboxylase was investigated using the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS). The binding of ANS to the enzyme molecule caused a significant increase of fluorescence emission which was further enhanced by the addition of Mg2+. The temperature dependence of the fluorescence emission indicated a conformational change of the enzyme between 12 and 24 degrees C. The Mg2+ and the temperature effects were additive. ANS itself did not change the conformation of the enzyme. The influence of the substrates carbon dioxide and ribulose-1,5-bisphosphate, and the effect of the pH of the medium and of a sulfhydryl reducing reagent on fluorescence emission were analysed.

The regulation of glucose-6-phosphate dehydrogenase in chloroplasts.

Glucose-6-phosphate dehydrogenase from intact pea chloroplasts is partially membrane bound and inactivated upon illumination. The inhibitory effect of light can be abolished by addition of methylviologen. Kinetic experiments with glucose-6-phosphate dehydrogenase reveal that, in the dark, the enzyme activity is strongly inhibited by the accumulation of NADPH. The inhibition of NADPH can be reversed by the addition of excess NADP+. The non-Michaelis-Menten-type kinetics suggest that the enzyme is stringently regulated by the ratio of NADPH to NADP+ plus NADPH, i.e., the "reduction charge". These observations seem to indicate that in the light the inhibition of glucose-6-phosphate dehydrogenase is due to a high reduction charge, whereas in the dark the enzyme is controlled by the metabolic demand for reducing equivalents.