Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.)

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg²⁺ ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a K_m of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO₃⁻ with S_0.5 value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, α-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca²⁺ and Mn²⁺. Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate.     

Malate inhibition of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate²⁻ concentration. Of the reaction components only HCO₃⁻ increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K_m, but no effect on V_max. Enzyme preincubated with malate shows the same effect of malate on K_m, but in addition V_max is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate.     

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase

The effect of 5-5′-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V_max is found to be independent of the presence of this thiol reagent. The K_m is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K_m (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K_m for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.     

pH Effects on the Activity and Regulation of the NAD Malic Enzyme

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg²⁺ and NAD⁺ but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate⁻². This is characteristic of an induced conformational change. The slope of the V_max or V_max/K_m profiles is steeper on the alkaline side of the pH optimum. The K_m for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K_{m pH6.7} = 9.2 millimolar, K_{m pH7.7} = 28.3 millimolar). Similar changes in K_m have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V_max/K_m profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.     

Regulation of Phosphoenolpyruvate Carboxylase from Crassula argentea: Further Evidence on the Dimer-Tetramer Interconversion

Phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants during the day exists in dimeric form the activity of which is strongly inhibited by malate. Enzyme purified from Crassula leaves collected during the day and stored at −70°C for 49 days shows a steady progression of change from dimer to tetramer, and this change in oligomeric state is accompanied by a decrease in the sensitivity of the enzyme to inhibition by malate. At 10 minutes preincubation of enzyme after 11 days storage—which is composed of an equilibrium mixture of dimer and tetramer—with malate causes most of the enzyme to be converted to dimer and increases the sensitivity of the enzyme to malate inhibition during assay. Preincubation with phosphoenolpyruvate shifts the equilibrium toward the tetrameric form and reduces the maximal inhibition produced by 5 millimolar malate to less than 20%. However, none of the treatments used resulted in shifting the oligomerization equilibrium completely in either direction. Thus the question of whether some covalent modification of the enzyme, such as phosphorylation, is required to permit complete changes in equilibrium remains open.     

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme : A Possible Regulatory Mechanism

NAD malic enzyme can exist in dimer, tetramer, or octamer form. Freshly prepared enzyme from Solanum tuberosum var. Chieftan exists predominantly as the octamer and during storage is progressively converted into lower molecular weight forms. High ionic strength favors dimer formation, whereas high concentrations of malate or citrate favor tetramer formation. The tetramer is the most active form, having a low K_m for malate and a high V_max. The dimer, with its high K_m and low V_max, is the least active form. Malate may regulate NAD malic enzyme by controlling its state of oligomerization.     

Light modulation of maize leaf phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH₄)₂SO₄ fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.     

Maize leaf phosphoenolpyruvate carboxylase : oligomeric state and activity in the presence of glycerol

Maize (Zea mays L.) leaf phosphoenopyruvate (PEP) carboxylase activity at subsaturating levels of PEP was increased by the inclusion of glycerol (20%, v/v) in the assay medium. The extent of activation was dependent on H⁺ concentration, being more marked at pH 7 (with activities 100% higher than in aqueous medium) than at pH 8 (20% activation). The determination of the substrate concentration necessary to achieve half-maximal enzyme activity (S_0.5) (PEP) and maximal velocity (V) between pH 6.9 and 8.2 showed a uniform decrease in S_0.5 in the presence of glycerol over the entire pH range tested, and only a slight decrease in V at pH values near 8. Including NaCl (100 millimolar) in the glycerol containing assay medium resulted in additional activation, mainly due to an increase in V over the entire range of pH. Glucose-6-phosphate (5 millimolar) activated both the native and the glycerol-treated enzyme almost to the same extent, at pH 7 and 1 millimolar PEP. Inhibition by 5 millimolar malate at pH 7 and subsaturating PEP was considerably lower in the presence of glycerol than in an aqueous medium (8% against 25%, respectively). Size-exclusion high performance liquid chromatography in aqueous buffer revealed the existence of an equilibrium between the tetrameric and dimeric enzyme forms, which is displaced to the tetramer as the pH was increased from 7 to 8. In the presence of glycerol, only the 400 kilodalton tetrameric form was observed at pH 7 or 8. However, dissociation into dimers by NaCl could not be prevented by the polyol. We conclude that the control of the aggregation state by the metabolic status of the cell could be one regulatory mechanism of PEP carboxylase.     

Kinetic characterization of spinach leaf sucrose-phosphate synthase

The spinach (Spinacia oleracea) leaf sucrose-phosphate synthase was partially purified via DEAE-cellulose chromatography, and its kinetic properties were studied. Fructose-6-phosphate saturation curves were sigmoidal, while UDPglucose saturation curves were hyperbolic. At subsaturating concentrations of fructose-6-phosphate, 1,5 anhydroglucitol-6-phosphate had a stimulatory effect on enzyme activity, suggesting multiple and interacting fructose-6-phosphate sites on sucrose-phosphate synthase. The concentrations required for 50% of maximal activity were 3.0 millimolar and 1.3 millimolar, respectively, for fructose-6-phosphate and UDPglucose. The enzyme was not stimulated by divalent cations. Inorganic phosphate proved to be a potent inhibitor, particularly at low concentrations of substrate. Phosphate inhibition was competitive with UDPglucose, and its K_i was determined to be 1.75 millimolar. Sucrose phosphate, the product of the reaction, was also shown to be a competitive inhibitor towards UDPglucose concentration and had K_i of 0.4 millimolar. The kinetic results suggest that spinach leaf sucrose-phospahte synthase is a regulatory enzyme and that its activity is modulated by the concentrations of phosphate, fructose-6-phosphate, and UDPglucose occurring in the cytoplasm of the leaf cell.     

Catalytic activity of maize leaf phosphoenolpyruvate carboxylase in relation to oligomerization

The relationship between the state of oligomerization and activity of purified maize leaf phosphoenolpyruvate carboxylase using size exclusion high performance liquid chromatography was examined. Maximum activities of 35 to 38 micromoles per minute per milligram protein were found when 100% of the enzyme was in its tetrameric form. The effects of the sulfhydryl group modifiers CuCl₂ and p-chloromercuribenzoate on enzyme inhibition and the state of aggregation of the protein complex were examined. Aggregation of the enzyme is temperature and pH sensitive with low temperature and high pH favoring depolymerization. Stability of the tetrameric form is largely dependent upon histidyl residues, and to some extent this explains the biphasic response of enzyme activity to changes in MgCl₂ concentrations. Modification of the tetramer's histidyl residues by the inhibitor diethylpyrocarbonate (0.125 millimolar) results in its dissociation to the dimeric form and loss of activity. Subsequent treatment with 0.4 molar hydroxylamine results in reassociation to the tetramer and restoration of enzymic activity.     

Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea

The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms.

The enzyme uses either Mg²⁺ or Mn²⁺ as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn²⁺ the K_m for malate²⁻ is lower than with Mg²⁺, but V_max is lower than with Mg²⁺. In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C₃ plants. In the reverse reaction, run with Mn²⁺, the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 × 10⁻³ molar.

The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this.

The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH.

The enzyme is activated by coenzyme A with K_a = 4 micromolar. AMP also shows competitive activation, with K_a = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate²⁻ is a strong activator, with K_a = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with K_i = 4.9 millimolar.

     

Occurrence of Two Pathways for Malate Oxidation in Bacteroids Isolated from Sesbania rostrata Stem Nodules during C(2)H(2) Reduction

Malate oxidation supported C₂H₂ reduction by bacteroids isolated from Sesbania rostrata stem nodules. Optimal activity reached 7.5 nanomoles per minute per milligram of dry weight and was in the same order of magnitude as that observed with succinate but always required a lower O₂ tension. Malate dehydrogenase (EC 1.1.1.37), purified 66-fold from bacteroids, actively oxidized malate (K_m = 0.19 millimolar). Malic enzyme (EC 1.1.1.39) from Sesbania bacteroids had a lower affinity for malate (K_m = 2.32 millimolar). Both enzymes exclusively required NAD⁺ as cofactor and required an alkaline pH for optimal activity. 2-Oxoglutarate and oxalate, inhibiting malate dehydrogenase and malic enzyme, respectively, were used to specifically block each malate oxidation pathway in bacteroids. The predominance of malate dehydrogenase activity to support bacteroid N₂ fixation was demonstrated. The inhibition of O₂ consumption by 2-oxoglutarate confirmed the importance of the malate dehydrogenase pathway in malate oxidation. It is proposed that the utilization of malate, with regard to O₂, is important in a general strategy of this legume to maintain N₂ fixation under O₂ limited conditions.     

Oligomerization and regulation of higher plant phosphoenolpyruvate carboxylase

The specific activity of phosphoenolpyruvate (PEP) measured at a saturating level of substrate diminishes as the enzyme is diluted at about the same rate that specific light scattering by the diluted enzyme decreases. The presence of PEP in the assay causes an increase in activity with increasing dilution. This is accompanied by an increase in light scattering of the diluted enzyme. The reverse situation obtains with the addition of malate to assays: the activity decreases with increasing dilution but light scattering is not substantially changed, indicating that the enzyme is already brought to a smaller aggregate by the dilution itself. In this case, the inhibition by malate in the assay probably is the noncompetitive type not involved in regulatory control by malate. Glucose-6-phosphate in the range from 1 to 6 millimolar causes an increase in activity of the enzyme run at a substrate level less than K_m, and an associated increase in light scattering is found, indicating an increase in the mean size of the enzyme. When PEP is added to a 1/80 diluted enzyme, light scattering increases and is associated with a more rapid activity of the enzyme. When malate is added to the same cuvette, the activity decreases and the light scattering diminishes, thus showing that the ligand response is immediately reversible. When malate is added first, followed by PEP, the reverse sequence of activity and light scattering change is observed.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Pyrophosphorylases in Solanum tuberosum: III. PURIFICATION, PHYSICAL, AND CATALYTIC PROPERTIES OF ADPGLUCOSE PYROPHOSPHORYLASE IN POTATOES

ADPglucose pyrophosphorylase from potato (Solanum tuberosum L.) tubers has been purified by hydrophobic chromatography on 3 aminopropyl-sepharose (Seph-C₃-NH₂). The purified preparation showed two closely associated protein-staining bands that coincided with enzyme activity stains. Only one major protein staining band was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The subunit molecular weight was determined to be 50,000. The molecular weight of the native enzyme was determined to be 200,000. The enzyme appeared to be a tetramer consisting of subunits of the same molecular weight. The subunit molecular weight of the enzyme is compared with previously reported subunit molecular weights of ADPglucose pyrophosphorylases from spinach leaf, maize endosperm, and various bacteria. ADPglucose synthesis from ATP and glucose 1-P is almost completely dependent on the presence of 3-P-glycerate and is inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg²⁺ are reported. The enzyme V_max is stimulated about 1.5- to 3-fold by 3 millimolar DTT. The significance of the activation by 3-P-glycerate and inhibition by inorganic phosphate ADPglucose synthesis catalyzed by the potato tuber enzyme is discussed.     

The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K_m (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K_i of about 6 millimolar. For comparison the K_i for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K_a values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and β-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.     

Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors

Glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the V_max value. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26s⁻¹ (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s⁻¹ (as a tetramer), the recycling rate of the active NAD moiety is 0.54 s⁻¹, and the apparent K_m value for glucose is 24 mM. Kinetically, lactate dehydrogenase behaves like a substrate with an apparent K_m value of 1.8 units·ml⁻¹ in this coupled reaction system with low coenzyme concentration. l-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.     

Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer–tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔC_p, with NADP binding ΔC_p closer to what is expected of crystallographic structures, while NADPH ΔC_p is three times larger. The estimated dimer–tetramer association constant is 1.5 · 10⁶ M^? 1, and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.     

Occurrence and Regulatory Properties of Uridine Diphosphatase in Fully Expanded Leaves of Soybean (Glycine max Merr.) and Other Species

High activities (100-200 micromoles UDP hydrolyzed per milligram chlorophyll per hour) of uridine-5′ diphosphatase (UDPase) have been identified in extracts of fully expanded soybean (Glycine max Merr.) leaves. In desalted crude extracts, UDPase activity was strongly inhibited by low concentrations of Mg:ATP (I₅₀ = 0.3 millimolar). Two forms of the enzyme were resolved by gel filtration on Sephadex G-150. The higher molecular weight form (UDPase I, about 199 kilodaltons by gel filtration) retained ATP sensitivity (I₅₀ = 0.3 millimolar), whereas the major, lower molecular weight form (UDPase II, about 58 kilodaltons) was markedly less sensitive to ATP inhibition (I₅₀ = 2.7-3.0 millimolar). Subsequent purification of UDPase I by ion-exchange chromatography on DEAE cellulose produced a lower molecular weight enzyme (about 74 kilodaltons by gel filtration) that had reduced ATP sensitivity similar to UDPase II. Ion-exchange chromatography of UDPase II did not alter molecular weight or ATP sensitivity. UDPase II, after the DEAE-cellulose step, was specific for nucleoside diphosphates. Maximum reaction velocity decreased in the following sequence; UDP > GDP > CDP. ADP was not a substrate for the enzyme. The reaction catalyzed was hydrolysis of the terminal-P of UDP to form UMP. The enzyme was stimulated by Mg²⁺ and the pH optimum was centered between pH 6.5 and 7.0. In a survey of various species, soybean cultivars had highest activities of apparent UDPase and other species ranged in apparent activity from 0 to 30 micromoles hydrolyzed per milligram chlorophyll per hour.     

Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases

Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C₄ enzyme to inhibition by malate is also unaffected by preincubation with these ligands.