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PubMed1984

Influence of glycerate on photosynthesis by wheat chloroplasts.

Glycerate was found to effect photosynthetic O2 evolution in wheat chloroplasts by its conversion to triose phosphate and by influencing the rate of photosynthesis through the reductive pentose phosphate pathway. In the absence of bicarbonate, the photosynthetic O2 evolution with glycerate was low (10 to 25 mumol mg chlorophyll-1 h-1), and only about 15% of the rate of bicarbonate-dependent O2 evolution under optimum conditions. This corresponds to a rate of glycerate conversion to triose phosphate of 20 to 50 mumol mg chlorophyll-1 h-1, which appears sufficient to accommodate flux through the glycolate pathway in vivo. Pi was required for this glycerate-dependent O2 evolution; rates remained relatively constant between 0.1 and 40 mM Pi, and proceeded with little lag upon illumination (less than 0.5 min). Evidence for O2 evolution due to glycerate conversion to triose phosphate could be conclusively demonstrated by addition of glycolaldehyde, an inhibitor of the regenerative phase of photosynthesis, which prevents CO2 fixation. The effect of glycerate on photosynthesis in the presence of bicarbonate was determined by measuring both photosynthetic O2 evolution and 14CO2 fixation at varying Pi concentrations. Low concentrations of glycerate (micro- to millimolar levels) prevented inhibition of photosynthesis by Pi. With 1 mM bicarbonate and pH 8.2, which is favorable for glycolate synthesis, maximum rates of photosynthesis were obtained at low Pi (25 microM), whereas strong inhibition of photosynthesis occurred at only 0.2 mM Pi. Addition of glycerate relieved the inhibition of photosynthesis by Pi, indicating the possible importance of glycerate metabolism in the chloroplast under photorespiratory conditions. The initiation of photosynthesis by glycerate at inhibitory Pi levels occurred with little reduction in the ratio of CO2 fixed/O2 evolved, and the main effect of glycerate was on carbon assimilation. While the basis for the beneficial effect of glycerate on CO2 assimilation under moderate to high Pi levels is uncertain, it may increase the concentration of 3-phosphoglycerate (PGA) in the chloroplast, and thus make conditions more favorable for induction of photosynthesis and reduction of PGA to triose phosphate.

Bicarbonates↗
PubMed1985

The substrate specificity, kinetics, and mechanism of glycerate-3-kinase from spinach leaves.

Glycerate-3-kinase (EC 2.7.1.31) from spinach leaves shows absolute specificity for D-glycerate as phosphate acceptor, yielding 3-phosphoglycerate as a product. ATP complexed with either Mg2+ or Mn2+ is the preferred phosphate donor. The enzyme has Km (D-glycerate) = 0.25 mM, Km (Mg-ATP) = 0.21 mM, Vmax = 300 mumol min-1 mg protein-1, and a turnover number = 12,000 X min-1. The equilibrium constant for the reaction is approximately 300 at pH 7.8. Pyrophosphate, 3-phosphoglycerate and ribulose 1,5-bisphosphate are the strongest inhibitors among the phosphorylated and nonphosphorylated metabolites tested; however, their regulatory role in vivo is questioned. Substrate kinetics, as well as product and analog inhibition data, are consistent with a sequential random mechanism. The distinct characteristic of the glycerate kinase-catalyzed reaction is the formation of a dead-end complex between the enzyme, D-glycerate, and 3-phosphoglycerate.

Adenosine Triphosphate↗
PubMed1986

Conversion of serine to glycerate in intact spinach leaf peroxisomes: role of malate dehydrogenase.

In photorespiration, leaf peroxisomes convert serine to glycerate via serine-glyoxylate aminotransferase and NADH-hydroxypyruvate reductase. We isolated intact spinach leaf peroxisomes in 0.25 M sucrose, and characterized their enzymatic conversion of serine to glycerate using physiological concentrations of substrates and coenzymes. In the presence of glycolate (glyoxylate), and NADH and NAD alone or together in physiological proportions, the rate of serine-to-glycerate conversion was enhanced and sustained by the addition of malate. The rate was similar at 1 and 5 mM serine, but was two to three times higher in 50 mM than 5 mM malate. In the presence of NAD and malate, there was 1:1 stoichiometric formation of glycerate and oxaloacetate. Addition of 1 or 5 mM glutamate resulted in a negligible enhancement of the conversion of hydroxypyruvate to glycerate. Intact peroxisomes produced glycerate from either serine or hydroxypyruvate at a rate two times higher than osmotically lysed peroxisomes. These results suggest that under physiological conditions, the peroxisomal malate dehydrogenase operates independent of aspartate-alpha-ketoglutarate aminotransferase in supplying NADH for hydroxypyruvate reduction. This supply of NADH is the rate-limiting step in the conversion of serine to glycerate. The compartmentation of hydroxypyruvate reductase and malate dehydrogenase in the peroxisomes confers a higher efficiency in the supply of NADH for hydroxypyruvate reduction under a normal, high NAD/NADH ratio in the cytosol.

Glyceric Acids↗
PubMed1988

Purification and kinetic and structural properties of spinach leaf NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase.

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2',5'-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 mumol (mg protein)-1 min-1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP+ resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP+. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min-1. D-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1-14C]iodoacetamide/mol enzyme. Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.

Ammonium Sulfate↗
PubMed1989

Coregulation of electron transport and Benson-Calvin cycle activity in isolated spinach chloroplasts: studies on glycerate 3-phosphate reduction.

Glycerate 3-phosphate-dependent O2 evolution was measured in intact chloroplasts in the absence of CO2. At all concentrations of added glycerate 3-phosphate oxygen evolution ceased before stoichiometric amounts of oxygen were evolved. The inhibition of glycerate 3-phosphate-dependent-O2 evolution increased with increasing concentrations of substrate added. A similar response was observed in chloroplasts treated with KCN which inhibits ribulose-1,5-bisphosphate carboxylase-oxygenase. Oxygen uptake via the oxygenase activity of this enzyme is therefore not the cause of the discrepancy in stoichiometry of oxygen release in this system. The addition of NaHCO3 to chloroplasts in which oxygen evolution was inhibited by glycerate 3-phosphate caused an immediate sustained rate of oxygen evolution in the absence of KCN but not with KCN present. Simultaneous measurements of chlorophyll a fluorescence showed that qQ remained oxidized, although net O2 evolution had ceased. As O2 evolution decreased, qE and delta pH increased. Upon the addition of the NaHCO3, QA became more oxidized while delta pH and qE were decreased, suggesting that the inhibition of electron transport at high glycerate 3-phosphate concentrations was mediated by photosynthetic control via delta pH. However, the levels of ATP, ADP, ribulose 1,5-bisphosphate, and Pi concentrations and ATP/ADP ratio. The stromal glycerate 3-phosphate content declined upon illumination until O2 evolution ceased. At this time a constant stromal glycerate 3-phosphate concentration of 8-10 mM was maintained while net import of glycerate 3-phosphate into the stroma had virtually ceased. The stromal triosephosphate content remained at a constant low level throughout but the glycerate 3-phosphate level increased slightly after addition of NaHCO3. The data provided by the measurements of thylakoid reactions and stromal metabolites suggest that photosynthetic electron transport is tightly coupled to the requirements of the stroma for ATP and NADPH. Glycerate 3-phosphate reduction requires much less ATP than the operation of the complete Benson-Calvin cycle since the stoichiometry of ATP and NADPH utilization is reduced to 1:1. We conclude that thylakoid electron flow is not sufficiently flexible to maintain NADPH and ATP production in the ratio of 1:1. This situation will favor overenergization of the thylakoid membrane, increased leakiness of protons, increased electron drainage to O2, and result in progressive inhibition of noncyclic electron flow.

Antimycin A↗
PubMed1989

The interaction of 3-phosphoglycerate and other substrate analogs with the glyoxylate- and succinate-binding sites of isocitrate lyase.

The gene for isocitrate lyase from Escherichia coli has recently been cloned and sequenced. However, knowledge of this enzyme from E. coli is limited. Because of the possible role of 3-phosphoglycerate as a metabolic inhibitor of isocitrate lyase in E. coli, a detailed analysis of this compound as an inhibitor is reported in this paper. Kinetic data suggest that 3-phosphoglycerate is an analog of isocitrate (or glyoxylate) and also that it competes with succinate, or succinate analogs, by interfering with their binding to the enzyme. This could be due to the steric bulk of the phosphate moiety of 3-phosphoglycerate extending in the direction of and over the succinate-binding site. The interaction of other substrate analogs, including glycolate, oxalate, phosphoenolpyruvate, and cis-aconitate, with isocitrate lyase from E. coli is also characterized.

Binding Sites↗
PubMed1992

25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase.

25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes.

Adenosine Triphosphate↗
PubMed1975

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark.

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 degrees C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3.Phosphoglycerate reduction, which is a partial reaction of CO2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO2 fixation without affecting phosphoglycerate reduction. This inhibition of CO2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO2 fixation in the light. This demonstrates that CO2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.

Adenosine Triphosphate↗
PubMed1977

The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro.

1. The formation of metabolites in the stroma compartment of isolated chloroplasts during carbon fixation, and their export to the medium, have been investigated using improved techniques. 2. Rapid separation of photosynthesising chloroplasts from the medium, accompanied by simultaneous quenching of metabolism was achieved by using silicone oil layer filtering centrifugation under illumination. Metabolites were separated by microscale ion-exchange chromatography. Quantitative determination of each metabolite was based on labelling with 32P. 3. It was found that fixed carbon was exported from the chloroplasts only as triose phosphate and phosphoglycerate, and to a minor extent, as pentose monophosphate. The main compounds accumulating in the stroma were hexose and heptose monophosphates and phosphoglycerate. A marked decrease in the concentration of inorganic phosphate in the stroma during the first 5 min of illumination was accompanied by a complementary increase in organic phosphate so that the total amount of phosphate within the chloroplasts remained constant. 4. The concentration difference for phosphoglycerate between the stroma and the medium was much higher than for triose phosphate or inorganic phosphate, although all three compounds are transported across the inner membrane of the chloroplast envelope by the same carrier. It was concluded that the efflux of phosphoglycerate was restricted.

Adenosine Triphosphate↗
PubMed1980

Relative activities of linear and cyclic electron flows during chloroplast CO2-fixation.

Evolution of oxygen and turnover of cytochromes b-563 and f were measured upon illumination of isolated intact spinach chloroplasts with a series of flashes. The flash yield of cytochrome f oxidation approximated the sum of the yields of cytochrome b-563 reduction and electron transfer through Photosystem II, regardless of whether HCO(-3), 3-phosphoglycerate or O2 served as the terminal electron acceptor. No absorbance contribution form cytochrome b-559 was discerned within the time range studied. Some pseudocyclic electron flow occurred when both HCO(-3) and 3-phosphoglycerate were omitted, and possibly also during induction of photosynthesis; however, the flash yield data suggest tht O2 is not reduced at a significant rate during steady state photosynthesis. The maximum rate of cytochrome f turnover (1000 microequiv./mg chlorophyll per h) was adequate to support the highest rates of photosynthesis observed in isolated chloroplasts. These results agree with the concept that cytochrome f is a component both of the linear and cyclic pathways whereas cytochrome b-563 functions only in the cyclic pathway. NH4Cl decreased the half time of cytochrome b-563 oxidation fro 11.6 to 8.2 ms and decreased the half time of cytochrome f reduction from 7.2 to 2.8 ms. The cyclic and linear pathways thus seem to be jointly regulated by a transthylakoid H+ gradient through a common control point on the reducing side of cytochrome f. Cyclic turnover also increased during the induction phase of photosynthesis, when linear throughput is limited by the rate of utilization of NADPH. The slow rise in the P-518 transient correlated with increased cyclic activity under the above conditions. It is proposed that flexibility in the utilization of linear and cyclic pathways allows the chloroplast to generate ATP and NADPH in ratios appropriate to varying needs.

Carbon Dioxide↗
PubMed1980

Effects of inorganic phosphate on the photosynthetic carbon reduction cycle in extracts from the stroma of pea chloroplasts.

1. Turnover of the photosynthetic carbon reduction cycle has been demonstrated in chlorophyll-free reaction mixtures containing chloroplast stromal extract, as evidenced by the fixation of CO2 following addition of small amounts of 3-phosphoglycerate. 2. The activity of the photosynthetic carbon reduction cycle in this system is inhibited by inorganic phosphate (Pi), with activity reduced to 50% by about 6.5 mM Pi. Pi also increased the lag period which elapsed before a steady rate of CO2 fixation was obtained. 3. The effect of Pi on the rate of 3-phosphoglycerate reduction following the addition of substrate amounts of some cycle intermediates was investigated. Substantial inhibition was observed with fructose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate and erythrose 4-phosphate as substrates. Pi also affected the activity of ribulose-bisphosphate carboxylase, with stimulation at Pi concentrations below 2.5 mM and inhibition at higher concentrations. 4. The results showed that the sedoheptulose bisphosphatase reaction is inhibited more strongly by Pi than the fructose bisphosphatase reaction. 5. It is concluded that the previously established inhibitory effects of Pi on photosynthesis by intact isolated chloroplasts may be partly due to these inhibitory effects of Pi on the reactions of the photosynthetic carbon reduction cycle.

Carbon Dioxide↗
PubMed1980

Energy charge, phosphorylation potential and proton motive force in chloroplasts.

Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M-1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M-1 in the dark and only 300 M-1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. delta G'ATP was -42 kJ/mol in darkened intact chloroplasts, -46 kJ/mol in illuminated intact chloroplasts and -60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH4Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO2 reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO2 reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force.

Adenosine Diphosphate↗