Addition Of 3-phosphoglycerate Research Articles

Domain motions in phosphoglycerate kinase: determination of interdomain distance distributions by site-specific labeling and time-resolved fluorescence energy transfer.

3-Phosphoglycerate kinase is composed of two globular domains separated by a wide cleft. The substrate binding sites are situated on the inner surfaces of the two domains. By analogy to other kinases, it has been postulated that the catalytic mechanism of phosphoglycerate kinase involves a hinge bending domain motion that brings the substrates together to allow phosphoryl transfer. To characterize this large-scale conformational change, as well as the dynamics of the unliganded enzyme in solution, we have applied site-directed mutagenesis and time-resolved nonradiative energy transfer techniques. Two genetically engineered cysteines (Cys-135 and Cys-290), one in each of the two domains, were covalently labeled with a donor and acceptor pair of fluorescent probes. Analysis of subnanosecond fluorescence decay curves yielded the equilibrium distribution of interdomain distances. In the absence of substrates, the distribution of distances between the two labeled sites was very broad, with a full width at half maximum estimated as 20 A or broader, indicative of a large number of conformational substates in solution. The mean distance, 31.5 +/- 1 A, was 8 A smaller than in the crystal structure. Upon addition of ATP alone or of ATP and 3-phosphoglycerate, the average distance increased to 38 +/- 1 A and the width of the distribution decreased. Addition of 3-phosphoglycerate alone induced a similar but smaller change. The rate of conformational state fluctuations (interconversion between states) was found to be slow on the nanosecond time scale, as expected for a protein with a relatively large interdomain contact area.

Contents of endogenous adenine nucleotides and ATPase activity of isolated whole chloroplasts

Endogenous adenine nucleotide contents of isolated intact chloroplasts and activities of the H +-ATPase were measured in parallel under various experimental conditions. In chloroplasts isolated from spinach grown in summer, ATPase activity declines upon light-dark transition in a biphasic manner. Initial rapid inactivation of about 50% of the ATPases corresponds with the rapid increase of the ADP and decrease of the ATP levels. The residual ATPases are slowly deactivated, probably due to the reversal of thiol modulation. In isolated chloroplasts from winter plants the phase of rapid inactivation is largely absent. Those chloroplasts have a low content of endogenous nucleotides. When the ratio of internal ATP ADP is decreased by the addition of 3-phosphoglycerate or the uncoupler FCCP, the activity of the ATPase decreases. The relationship between activity and ATP ADP ratio is nearly linear. Inorganic phosphate, although being ineffective in changing the internal nucleotide levels, significantly raises the ATPase activity. The results suggest that in whole chloroplasts the ATPase not only is regulated by the thioredoxin system but also controlled by the internal ATP ADP ratio. This kind of control is referred to the nucleotide-dependent regulatory mechanism previously demonstrated in isolated thylakoid suspension.

Energy-dependent activation of spore-lytic enzyme precursor by germinated spores of [formula omitted

A precursor of the spore-lytic enzyme of Clostridium perfringens was extracted with alkali from dormant spores of the organism. The enzyme precursor was activated by incubating it with germinated spores which had been treated with alkali. The activation was greatly enhanced by the addition of 3-phosphoglycerate, suggesting that the conversion of precursor to active enzyme depends on endogenous energy-producing metabolism during germination.

Slow secondary fluorescence kinetics associated with the onset of photosynthetic carbon assimilation in intact isolated chloroplasts

Experiments with carefully isolated, largely intact chloroplasts, capable of fast rates of CO 2 -dependent O 2 evolution, show that the fall in chlorophyll a fluorescence (from the early maxima reached immediately after illumination) is interrupted by a ‘shoulder’ which is associated with the exponential increase in the rate of O 2 evolution. The length of this induction period was increased by storage, by decreased temperature, by increased orthophosphate concentration in the assay medium or by the presence of D, L-glyceraldehyde. It could also be shortened by the addition of 3-phosphoglycerate or dihydroxyacetonephosphate. In each treatment the shoulder in fluorescence shifted so that the association with the period of exponential increase was maintained. When illumination was re-started after a short dark interval, induction was minimal and no shoulder could be discerned, but both the lag in the onset of O 2 evolution and the shoulder were restored when the chloroplasts were resuspended in fresh assay medium during the period of darkness. The relation between chlorophyll a fluorescence and the onset of photosynthetic carbon assimilation is discussed.

Modulation of NADP-Malate Dehydrogenase Activity in Maize Mesophyll Chloroplasts

When intact mesophyll chloroplasts of Zea mays var Kelvedon Glory were illuminated, activation of NADP-malate dehydrogenase occurred. Activity declined rapidly on darkening. Light activation of the enzyme was very much greater in the presence of pyruvate ( approximately 10- to 20-fold) than with the electron acceptors 3-phosphoglycerate or oxaloacetate present ( approximately 2-fold). Following preillumination in the presence of pyruvate, addition of 3-phosphoglycerate, oxaloacetate, or nitrite substantially diminished the activity of NADP-malate dehydrogenase. In these circumstances, with pyruvate and 3-phosphoglycerate present, activity could be restored by the addition of nigericin or dihydroxyacetone phosphate. Nigericin also restored activity with both oxaloacetate and pyruvate present. The effect of nitrite was more marked in the presence of low concentrations of DCMU.These observations are discussed in terms of the dependence of enzyme activity upon the redox state of ferredoxin and electron carriers; the redox state of the latter was estimated by analysis of the DCMU-induced relaxation kinetics of chlorophyll fluorescence quenching in the presence of different substrates.

Relationships between protein phosphorylation and electron transport in the reconstituted chloroplast system.

Phosphorylation of the light-harvesting chlorophyll protein (LHCP) by the thylakoid protein kinase has been examined in the reconstituted chloroplast system. The level of phosphorylation by [32P]Pi was maximal at high light intensity and in the absence of 3-phosphoglycerate; dephosphorylation resulted from a subsequent decrease in light intensity or from the addition of 3-phosphoglycerate. Addition of ribose 5-phosphate, which acts as an ATP 'sink', also caused dephosphorylation. It is concluded that the degree of phosphorylation is dependent on the redox state and energy state of the system, thereby providing a mechanism for adapting light harvesting to the demands of carbon assimilation.

C 4 acid decarboxylation and CO 2 donation to photosynthesis in bundle sheath strands and chloroplasts from species representing three groups of C 4 plants

The mechanism of decarboxylation of C 4 acids and carboxyl donation to the Calvin pathway was studied in bundle sheath strands and chloroplasts isolated from leaves of species representing three groups of C 4 plants: Digitaria sanguinalis (NADP-malic enzyme type), Panicum miliaceum (NAD-malic enzyme type), and Eriochloa borumensis (phosphoenolpyruvate-carboxykinase type). High rates of 14CO 2 fixation and HCO 3− dependent O 2 evolution were exhibited by bundle sheath strands and chloroplasts of P. miliaceum and E. borumensis, while those of D. sanguinalis (the NADP-malic enzyme type) required the addition of ribose 5-phosphate and malate for maximum activity. In all three species, 3-(3-4-dichlorophenyl)-1,1-dimethylurea inhibition of 14CO 2 fixation was partially overcome by the addition of malate, suggesting that malate can serve as a source of reductive power for carbon assimilation. Decarboxylation of 4- 14C-labeled C 4 acids was followed in the presence of glyceraldehyde, an inhibitor of the Calvin pathway. A large light-dependent stimulation of malate decarboxylation occurred by the addition of 3-phosphoglycerate (6 m m) with bundle sheath cells of all three groups and with bundle sheath chloroplasts of D. sanguinalis and E. borumensis. Although malate is suggested to be decarboxylated through the respective primary decarboxylase of each species, the 3-phosphoglycerate enhancement suggests that pyridine nucleotide reduction from malate oxidation is used during the reductive phase of the Calvin pathway. Bundle sheath strands from all three species decarboxylated aspartate with severalfold stimulation by the addition of either α-ketoglutarate (10 m m) or pyruvate (1 m m), with the lowest rate in D. sanguinalis. Bundle sheath chloroplasts from either of the three species could not decarboxylate aspartate. A light-independent decarboxylation of aspartate is suggested through NAD-malic enzyme in bundle sheath mitochondria in all three types of C 4 species, the rates being highest in P. miliaceum. A light-dependent portion of aspartate decarboxylation in E. borumensis is suggested to be through phosphoenolpyruvate carboxykinase. C 4. acid decarboxylation in all three species was inhibited by HCO 3− (CO 2 may be the active species). Consistent with the C 4 acid decarboxylation studies, the bundle sheath strands of the three species gave light-dependent O 2 evolution with aspartate or malate, the bundle sheath chloroplasts of D. sanguinalis and E. borumensis exhibited light-dependent O 2 evolution only with malate, and the bundle sheath chloroplasts of P. miliaceum showed no C 4 acid-dependent O 2 evolution. In all three C 4 types, malate decarboxylation, though by different decarboxylases, interacted with the Calvin pathway by providing both CO 2 and reducing power, while aspartate decarboxylation provided only CO 2.

Magnetic resonance studies on manganese-nucleotide complexes of phosphoglycerate kinase

Measurements of the relaxation rate of water protons (PRR) have been used to study the interaction of yeast phosphoglycerate kinase with the manganous complexes of a number of nucleotides. The results indicate that phosphoglycerate kinase belongs to the same class of enzymes as creatine kinase, adenylate kinase, formyltetrahydrofolate synthetase, and arginine kinase, with maximal binding of metal ion to tne enzyme in the presence of the nucleotide substrate. However, an analysis of titration curves for a number of nucleoside diphosphates (ADP, IDP, GDP) showed that there is a substantial synergism in binding of the metal ion and nucleotide to the enzyme in the ternary complex. The metal-substrate binds to the enzyme approximately two orders of magnitude more tightly than the free nucleotide; Other evidence for an atypical binding scheme for Mn(II)-nucleoside diphosphates was obtained by electron paramagnetic resonance (EPR) studies; the EPR spectrum for the bound Mn(II) in the enzyme-MnADP complex differed substantially from those obtained for other kinases. An identical EPR spectrum is observed with the MnADP complex with the rabbit muscle enzyme as with the yeast enzyme. In contrast, the dissociation constant for the enzyme-MnATP complex is approximately fourfold lower than that for enzyme-ATP, and there are no substantial changes in the electron paramagnetic resonance spectrum of MnATP2- when the complex is bound to phosphoglycerate kinase. A small but significant change in the PRR of water is observed on addition of 3-phosphoglycerate (but not 2-phosphoglycerate) to the MnADP-enzyme complex. However, addition of 3-phosphoglycerate to enzyme-MnADP did not influence the EPR spectrum of the enzyme-bound Mn(II).

The effect of dihydroxyacetone phosphate and 3-phosphoglycerate on O 2 evolution and on the levels of ATP, ADP and P i in isolated intact chloroplasts

Addition of dihydroxyacetone phosphate (2.5 mM) or 3-phosphoglycerate (2.5 mM) to a suspension of isolated intact chloroplasts, which contains P i only in low concentrations (0.2 mM) leads to a competitive inhibition of P i uptake in the light. In consequence, the ATP/ADP ratio is strongly decreased. The rate of O 2 evolution is also reduced under these conditions, but the degree of inhibition is much higher after addition of dihydroxyacetone phosphate than after addition of 3-phosphoglycerate. Therefore, besides the competitive inhibition of P i uptake, additional effects of dihydroxyacetone phosphate and 3-phosphoglycerate on O 2 evolution and CO 2 fixation of isolated intact chloroplasts must occur, which are discussed.

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO 2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ⩾ 2.5 mM completely inhibits CO 2-dependent O 2 evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P i, but not by addition of 3-phosphoglycerate. In the absence of P i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the 14C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O 2 evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts. 2. Although O 2 evolution is completely suppressed by dihydroxyacetone phosphate, CO 2 fixation takes place in air with rates of up to 65μ mol · mg −1 chlorophyll · h −1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation. 3. Under anaerobic conditions, the rates of CO 2 fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg −1 chlorophyll · h −1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10 −7 M) reaching values of up to 60 μmol · mg −1 chlorophyll · h −1. As under these conditions the ATP necessary for CO 2 fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO 2 fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10 −7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO 2 fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO 2 fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation. 4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO 2 fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.

Carbon dioxide fixation by isolated chloroplasts of Euglena gracilis: I. Isolation of functionally intact chloroplasts and their characterization

A method was developed for isolating functionally intact chloroplasts from heterotrophically grown Euglena. These isolated chloroplasts are capable of fixing CO 2 at rates up to 30 μmoles/mg chlorophyll/hr, which is one-half the rate of the whole cells. In contrast to chloroplasts isolated from spinach, Euglena chloroplasts are strongly inhibited by the presence of sucrose or by isolation at low pH. ATP is not able to reverse the inhibition of CO 2 fixation by uncouplers of photophosphorylation, indicating that the chloroplast envelope is not permeable to ATP. Pyrophosphate stimulates CO 2 fixation only in the absence of inorganic phosphate. The presence of inorganic phosphate causes a lag period before maximum rates of CO 2 fixation are achieved, and this lag is diminished by the addition of 3-phosphoglycerate and ribose-5-phosphate. Pyocyanin inhibits CO 2 fixation, and the addition of ribose-5-phosphate fails to reverse this inhibition.

Some effects of inorganic phosphate on O 2 evolution by isolated chloroplasts

1. After an initial lag, isolated spinach chloroplasts evolved O 2 in illuminated reaction mixtures containing bicarbonate but no added phosphate. This evolution soon ceased but could be restarted by the addition of phosphate. 2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O 2 were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate. 3. With CO 2 as the sole added substrate the extent of the initial lag in O 2 evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO 2. 4. There was also a requirement for phosphate by chloroplasts evolving O 2 in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO 2. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O 2 evolution which normally followed the addition of 3-phosphoglycerate. 5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity.