Dissociation and characterization of enzymes from a multienzyme complex involved in CO2 fixation.

A multienzyme complex from Euglena, molecular weight about 360,000, containing phosphoenolpyruvate carboxylase, malate dehydrogenase, and acetyl-coenzyme A carboxylase has been dissociated into active constituent enzymes. The respective molecular weights are 183,000, 67,000, and 127,000. The malate dehydrogenase contained in the complex is electrophoretically distinct from other malate dehydrogenase isozymes found in Euglena. The K-m for HCO3minus of the free and complexed acetyl-CoA carboxylase is 4.2-5.4 mM, and the substrate dependency for acetyl-CoA describes a sigmoidal relationship. The HCO3minus K-m for the free phosphoenolpyruvate carboxylase is 7.3-5.4 mM while that for the same enzyme contained in the complex is 0.7-1.3 mM. Both the free and complexed forms ofphosphoenolpyruvate carboxylase have a K-m for phosphoenolpyruvate of 0.9-1.7 mM. The latter enzyme in both the complex and free forms is stimulated by NADH, acetyl-CoA, and ATP. In the free phosphoenolpyruvate carboxylase, the stimulation passes through a maximum depending on effector concentration. The effect of NADH is to increase V-max while K-m values remain unmodified.     

Purification and Characterization of 3-Methylcrotonyl-Coenzyme A Carboxylase from Higher Plant Mitochondria

3-Methylcrotonyl-coenzyme A (CoA) carboxylase was purified to homogeneity from pea (Pisum sativum L.) leaf and potato (Solanum tuberosum L.) tuber mitochondria. The native enzyme has an apparent molecular weight of 530,000 in pea leaf and 500,000 in potato tuber as measured by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate disclosed two nonidentical subunits. The larger subunit (B subunit) is biotinylated and has an apparent molecular weight of 76,000 in pea leaf and 74,000 in potato tuber. The smaller subunit (A subunit) is biotin free and has an apparent molecular weight of 54,000 in pea leaf and 53,000 in potato tuber. The biotin content of the enzyme is 1 mol/133,000 g of protein and 1 mol/128,000 g of protein in pea leaf and potato tuber, respectively. These values are consistent with an A4B4 tetrameric structure for the native enzyme. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8 to 8.3 and at 35 to 38[deg]C in the presence of Mg2+. Kinetic constants (apparent Km values) for the enzyme substrates 3-methylcrotonyl-CoA, ATP, and HCO3- were: 0.1 mM, 0.1 mM, and 0.9 mM, respectively, for pea leaf 3-methylcrotonyl-CoA carboxylase and 0.1 mM, 0.07 mM, and 0.34 mM, respectively, for potato tuber 3-methylcrotonyl-CoA carboxylase. A steady-state kinetic analysis of the carboxylase-catalyzed carboxylation of 3-methylcrotonyl-CoA gave rise to parallel line patterns in double reciprocal plots of initial velocity with the substrate pairs 3-methylcrotonyl-CoA plus ATP and 3-methylcrotonyl-CoA plus HCO3- and an intersecting line pattern with the substrate pair HCO3- plus ATP. It was concluded that the kinetic mechanism involves a double displacement. Purified 3-methylcrotonyl-CoA carboxylase was inhibited by end products of the reaction catalyzed, namely ADP and orthophosphate, and by 3-hydroxy-3-methylglutaryl-CoA. Finally, as for the 3-methylcrotonyl-CoA carboxylases from mammalian and bacterial sources, plant 3-methylcrotonyl-CoA carboxylase was sensitive to sulfhydryl and arginyl reagents.     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Illumination increases the phosphorylation state of maize leaf phosphoenolpyruvate carboxylase by causing an increase in the activity of a protein kinase

Illumination of maize leaves increases the phosphorylation state of phosphoenolpyruvate carboxylase and reduces the sensitivity of the enzyme to feedback inhibition by malate. Red, white and blue light were each found to be equally potent, and the effect of light was blocked by 3(3,4-dichlorophenyl)-1,1-dimethylurea. A phosphoenolpyruvate carboxylase kinase was partially purified from illuminated maize leaves by a three-step procedure. Phosphorylation of phosphoenolpyruvate carboxylase by this protein kinase reached 0.7-0.8 molecules/subunit and correlated with a 3- to 4-fold increase in Ki for malate. The protein kinase was inhibited by L-malate, but was insensitive to a number of other potential regulators. Freshly prepared and desalted extracts of darkened maize leaves contained very little kinase activity, but the activity appeared when leaves were illuminated for 30-60 min before extraction. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle, but not that of protein phosphatase 1, could dephosphorylate phosphoenolpyruvate carboxylase. The protein phosphatases 1 and 2A activities of maize leaves were not affected by illumination. It is suggested that the major means by which light stimulates the phosphorylation of phosphoenolpyruvate carboxylase is by an increase in the activity of the protein kinase.     

Fractionation of stable carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants

The active species of "CO(2)" and the amount of fractionation of stable carbon isotopes have been determined for a partially purified preparation of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) from corn (Zea mays) leaves. The rates of the enzyme reactions, using substrate amounts of HCO(3) (-), CO(2) or CO(2) plus carbonic anhydrase, show that HCO(3) (-) is the active species of "CO(2)" utilized by PEP carboxylase. The K(m) values for CO(2) and HCO(3) (-) are 1.25 mm and 0.11 mm, respectively, which further suggest the preferential utilization of HCO(3) (-) by PEP carboxylase. The amount of fractionation of stable carbon isotopes by PEP carboxylase from an infinite pool of H(12)CO(3) (-) and H(13)CO(3) (-) was -2.03 per thousand. This enzyme fractionation (delta), together with the fractionation associated with absorption of CO(2) into plant cells and the equilibrium fractionation associated with atmospheric CO(2) and dissolved HCO(3) (-) are discussed in relation to the fractionation of stable carbon isotopes of atmospheric CO(2) during photosynthesis in C(4) plants.     

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase.

A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Purification, quaternary structure, composition, and properties of D-ribulose-1,5-bisphosphate carboxylase from Thiobacillus intermedius.

D-Ribulose-1,5-bisphosphate (RuBP) carboxylase has been purified from glutamate-CO2-S2O3(2)-grown Thiobacillus intermedius by pelleting the enzyme from the high-speed supernatant and by intermediary crystallization followed by sedimentation into a discontinuous 0.2 to 0.8 M sucrose gradient. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels of several acrylamide concentrations, sedimentation velocity and equilibrium measurements, and electron microscopic observations of negatively stained preparations. The molecular weights of the enzyme determined by sedimentation equilibrium and light-scattering measurements averaged 462,500 +/- 13,000. The enzyme consisted of closely similar or identical polypeptide chains of a molecular weight of 54,500 +/- 5,450 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The S(0)20,w of the enzyme was 18.07S +/- 0.22. Electron microscopic examination suggested that the octomeric enzyme (inferred from the molecular measurements mentioned) had a cubical structure. The specific activity of the enzyme was 2.76 mumol of RuBP-dependent CO2 fixed/min per mg of protein (at pH 8 and 30 C), and the turnover number in terms of moles of CO2 fixed per mole of catalytic site per second was 2.6. The enzyme was stable for 3 months at -20 C and at least 4 weeks at 0 C. The apparent Km for CO2 was 0.75 mM, and Km values for RuBP and Mg2+ were 0.076 and 3.6 mM, respectively. Dialyzed enzyme could be fully reactivated by the addition of 20 mM Mg2+ and partially reactivated by 20 mM Co2+, but Cd2+, Mn2+, Ca2+, and Zn2+ had no effect. The compound 6-phosphogluconate was a linear competitive inhibitor with respect to RuBP when it had been preincubated with enzyme, Mg2+, and HCO3-.     

Regulation of phosphoenolpyruvate carboxylase from maize leaves by nitrate and alanine.

Nitrate and alanine were found to stimulate partially purified maize leaf phosphoenolpyruvate carboxylase under specific assay conditions. Both metabolites stimulated the enzyme at low pH (7.0-7.5) and low substrate levels (1mM phosphoenolpyruvate). Nitrate was found to have a biphasic effect on the enzyme, stimulating at low concentrations (1mM-3mM), with a decrease in stimulation at higher levels. Nitrate caused inhibition of activity at pH 8.0 and although alanine caused some stimulation in activity at pH 8.0 this was not as marked as at the lower pH levels.     

The initial synthesis of proteins during development. Phosphoenolpyruvate carboxylase in rat liver at birth

1. A specific antibody, prepared by immunizing rabbits with phosphoenolpyruvate carboxylase (EC 4.1.1.32) purified from adult rat liver, was used to study the appearance of this enzyme in livers from developing rats. 2. Although some inactive precursor of the enzyme may be present in foetal liver, the amount is not sufficient to account for the enzyme appearance at birth. 3. The rate of phosphoenolpyruvate carboxylase synthesis relative to other cytosol proteins increases 20-fold from the foetus to the 1-day-old rat. The high rate of synthesis was maintained at least until 3 days after birth. 4. There was no measurable degradation of phosphoenolpyruvate carboxylase during the first day after birth. During this period the hepatic enzyme content increased 12-fold. 5. When phosphoenolpyruvate carboxylase attained a constant activity in the liver of rats 2 days after birth the half-time of degradation was approx. 13h. 6. We suggest that the pattern of changes occurring during appearance of phosphoenolpyruvate carboxylase is similar to substrate-induced enzyme induction in bacteria.     

Phosphoenolpyruvate carboxykinase activity in grape berries

Phosphoenolpyruvate (PEP) carboxykinase activity was found in crude extracts of ;Pinot noir' grape berries. The enzyme required ATP, Mn(2+) plus Mg(2+), a pH of 6.6, and a temperature of 40 C for maximum activity. The range in concentration of oxaloacetic acid needed for maximum phosphoenolpyruvate carboxykinase activity was 5 to 10 mm, and the Km for HCO(3) (-) in the exchange of (14)CO(2) into oxaloacetic acid was 26.8 mm.Changes in the activity of PEP carboxykinase and PEP carboxylase in berries were studied at weekly intervals throughout fruit development. PEP carboxykinase had maximum activity 4 weeks after flowering, and during the following 11 weeks remained relatively constant. The activity of PEP carboxylase was 2- to 4-fold higher than PEP carboxykinase throughout fruit development, and changed little except for a sharp reduction at the onset of ripening.     

ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.     

PURIFICATION OF RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE AND CARBON ISOTOPE FRACTIONATION BY WHOLE CELLS AND CARBOXYLASE FROM CYLINDROTHECA SP. (BACILLARIOPHYCEAE.)1,2,3

Ribulose 1,5-biphosphate carboxylase has been purified to homogeneity from extracts of Cylindrotheca sp. (strain N-1), a marine, pennate diatom. The carboxylase has a molecular weight and structural composition similar to the enzyme from higher plants. When assayed in the presence of 1 mM NaHCO₃ the enzyme was stimulated nearly 40% by 1 mM aspartate and over 20% by 1 mM malate, and was inhibited to over 60% by 1 mM phosphoenolpyruvate. Similar experiments, using spinach carboxylase, failed to show activation by these metabolites. When assayed in the presence of 20 mM NaHCO₃, 6-phosphogluconate (1 mM) inhibited activity of ribulose bisphosphate carboxylase from Cylindrotheca by 60%, and higher concentrations of maiate (10 mM) inhibited activity by 25% Carbon isotope fractionation by ribulose bisphosphate carboxylase was -32.6% (ppt) when measured under N₂ using homogeneous enzyme, whereas maximum carbon isotope fractionation by the whole alga grown in 1% -C0₂-in air averaged - 16.8%. Carbon isotope fractionation by the whole alga varied with the density of the culture and was maximum at a low cell density (1.7 ± 10⁶ cellslml). At higher densities, the fractionation decreased by 4.0%. Carbon isotope fractionation has been used previously to determine the pathway of carbon metabolism in other organisms; the results of this investigation seem to indicate that this strain uses both the reductive pentose phosphate pathway and the C₄ carbon pathway for primary CO₂ fixation.     

Phosphoenolpyruvate carboxylase of Thiobacillus thioparus. I. General properties.

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxylase (phosphorylating), EC 4.1.1.31) was purified 19-fold from the obligate chemoautotroph, Thiobacillus thioparus. Michaelis constants for the substrates were found to be 0.44 mM for phosphoenolpyruvate, 0.89 mM for bicarbonate, and 0.37 mM for magnesium, using Tris-HC1, pH 7.3. 1-Aspartate, 1-malate, and orthophosphate were found to be inhibitors of enzyme activity, while acetyl CoA, FDP, GTP, and CDP had no effect. Dioxane greatly stimulated enzyme activity.     

Inhibition of CA V decreases glucose synthesis from pyruvate

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.     

Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium.

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.     

Phosphoenolpyruvate carboxylase from the roots of yellow lupin (Lupinus luteus)

A crude preparation of PEP carboxylase (EC 4.1.1.31) from the yellow lupin roots exhibits the pH optimum of activity within the range of 7.4-8.6 and the temperature optimum at 32 - 40 degrees C. Its Km for PEP is 0.1 mM, and Km for HCO3- is 0.7 mM. The affinity of the enzyme towards Mg2+ diminishes with the metal ion concentration. At the concentration of Mg2+ below 0.5 mM Km for Mg2+ is 0.07 mM and at the Mg2+ concentration over 1.5 mM it rises to 0.47 mM. The Hill coefficients are 0.37 and 0.88, respectively. Among several compounds affecting the PEP carboxylase activity, such as organic acids, amino acids, and sugar phosphates, at physiological pH (7.0 and 7.8), malate shows the strongest inhibition of a competitive character, its Ki being 2 mM. Also acidic amino acids strongly inhibit the enzyme activity, aspartate being more effective than glutamate. Glucose 6-phosphate and fructose 1,6-diphosphate markedly activate the enzyme. Both the inhibition by malate, aspartate and glutamate, and the activation by sugar phosphates rises considerably when pH is decreased from 7.8 to 7.0. Malonate scarcely affects the enzyme.     

Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C4 plant, Amaranthus hypochondriacus

Illumination increased markedly the affinity to bicarbonate of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) in leaves of Amaranthus hypochondriacus L., a C4 plant. When leaves were illuminated, the apparent Km for (HCO3-) of PEPC decreased by about 50% concurrent with a 2- to 5-fold increase in Vmax and 3- to 4-fold increase in Ki for malate. The inclusion of ethoxyzolamide, an inhibitor of carbonic anhydrase, during the assay had no effect on kinetic and regulatory properties of PEPC indicating that carbonic anhydrase was not involved during light-induced sensitization of PEPC to HCO3-. Pretreatment of leaf discs with cycloheximide (CHX), a cytosolic protein synthesis inhibitor, suppressed significantly the light-enhanced decrease in apparent Km (HCO3-). Further, in vitro phosphorylation of purified dark-form PEPC by protein kinase A (PKA) decreased the apparent Km (HCO3-) of the enzyme, in addition increasing Ki (malate) as expected. Such changes, due to in vitro phosphorylation of purified PEPC by PKA, occurred only with wild-type PEPC, but not in the mutant form of maize (S15D) which is already a mimic of the phosphorylated enzyme. These results suggest that phosphorylation of the enzyme is important during the sensitization of PEPC to HCO3- by illumination in C4 leaves. Since illumination is expected to increase the cytosolic pH and the availability of dissolved HCO3- in mesophyll cells, the sensitization by light of PEPC to HCO3- could be physiologically quite significant.     

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12. 2. Kinetic properties.

1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.