Purification of Spinach Leaf ADPglucose Pyrophosphorylase

ADPglucose pyrophosphorylase from spinach leaves has been purified to homogeneity by hydrophobic chromatography carried out in 1 molar phosphate buffer. After polyacrylamide gel electrophoresis, the preparation showed only one protein staining band that coincided with a single activity stain. The enzyme appears to be composed of two subunits with molecular weights of 44,000 and 48,000, respectively, as determined by SDS polyacrylamide gel electrophoresis. Thus ADPglucose pyrophosphorylase of spinach appears to be comprised of subunits which are similar in size to the subunits of ADPglucose pyrophosphorylase isolated from bacterial sources. In contrast, a subunit molecular weight of 96,000 has been reported for the maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). The purified enzyme retains similar allosteric and catalytic properties as reported previously and is more sensitive to phosphate inhibition under “dark”-simulated conditions than under “light”-simulated conditions.     

Fructose 2,6-bisphosphate, carbohydrate partitioning, and crassulacean Acid metabolism

To initiate structural studies of the ADPglucose pyrophosphorylase from spinach an improved purification procedure was devised. The modified purification scheme allowed the isolation of 20 to 30 milligrams pure enzyme from 10 kilogram of spinach leaves. Electrophoresis of the purified enzyme confirmed an earlier study which showed that the enzyme was putatively composed of two subunits (Copeland L, J Preiss 1981 Plant Physiol 68: 996-1001). The two subunits migrate as 51 and 54 kilodalton proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both proteins can be detected on Western blots of leaf homogenates prepared under denaturing conditions suggesting that both subunits exist in vivo. Anion-exchange chromatography in the presence of urea allowed resolution of the 51 and 54 kilodalton proteins. They possess different N-terminal amino acid sequences and tryptic peptide maps. Western blot analysis reveals that the 51 and 54 kilodalton proteins are antigenically dissimilar. The 51 but not the 54 kilodalton protein is immunologically related to the ADPglucose pyrophosphorylase from maize endosperm and potato tuber.     

The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase

ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.     

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.     

Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases

ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purified from two cyanobacteria: the filamentous, heterocystic, Anabaena PCC 7120 and the unicellular Synechocystis PCC 6803. The purification procedure gave highly purified enzymes from both cynobacteria with specific activities of 134 (Synechocystis) and 111 (Anabaena) units per milligram protein. The purified enzymes migrated as a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular mass corresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons. Tetrameric structures were determined for the native enzymes by analysis of gel filtrations. Kinetic and regulatory properties were characterized for the cyanobacterial ADPglucose pyrophosphorylases. Inorganic phosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. The Synechocystis enzyme was activated 126-fold by 3-phosphoglycerate, with saturation curves exhibiting sigmoidicity (A_0.5 = 0.81 millimolar; n_H = 2.0). Activation by 3-phosphoglycerate of the enzyme from Anabaena demonstrated hyperbolic kinetics (A_0.5 = 0.12 millimolar; n_H = 1.0), having a maximal stimulation of 17-fold. I_0.5 values of 95 and 44 micromolar were calculated for the inhibition by inorganic phosphate of the Synechocystis and Anabaena enzyme, respectively. Pyridoxal-phosphate behaved as an activator of the cyanobacterial enzyme. It activated the enzyme from Synechocystis nearly 10-fold with high apparent affinity (A_0.5 = 10 micromolar; n_H = 1.8). Phenylglyoxal modified the cyanobacterial enzyme by inactivating the activity in the presence of 3-phosphoglycerate. Antibody neutralization experiments showed that anti-spinach leaf (but not anti-Escherichia coli) ADPglucose pyrophosphorylase serum inactivated the enzyme from cyanobacteria. When the cyanobacterial enzymes were resolved on sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoresis and probed with Western blots, only one protein band was recognized by the anti-spinach leaf serum. The same polypeptide strongly reacted with antiserum prepared against the smaller spinach leaf 51 kilodalton subunit, whereas the anti-54 kilodalton antibody raised against the spinach subunit reacted weakly to the cyanobacterial subunit. Regulatory and immunological properties of the cyanobacterial enzyme are more related to the higher plant than the bacterial enzyme. Despite this, results suggest that the ADPglucose pyrophosphorylase from cyanobacteria is homotetrameric in structure, in contrast to the reported heterotetrameric structures of the higher plant ADPglucose pyrophosphorylase.     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.     

A Starch Deficient Mutant of Arabidopsis thaliana with Low ADPglucose Pyrophosphorylase Activity Lacks One of the Two Subunits of the Enzyme

A starch deficient mutant of Arabidopsis thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild type. A single, nuclear mutation at a previously undescribed locus designated adg2 is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild type, it accumulated 40% as much starch when grown in a 12 hour photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates its steady state accumulation. Immunological analysis of leaf extracts using antibodies against the spinach 54 and 51 kilodalton (kD) ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54 kD polypeptide and has only about 4% as much as the wild type of a cross-reactive 51 kD polypeptide. This result and genetic studies suggested that adg2 is a structural gene which codes for the 54 kD polypeptide, and provides the first functional evidence that the 54 kD polypeptide is a required component of the native ADPglucose pyrophosphorylase enzyme.     

Biosynthesis of bacterial glycogen: Kinetic studies of a glucose-1-P adenylyltransferase (EC 2.7.7.27) from a glycogen-excess mutant of Escherichia coli B

An Escherichia coli B mutant, CL1136 accumulates glycogen at 3.4 to 4 times the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to α- and β-amylolysis, chain length determination and I₂-complex absorption spectra. The CL1136 mutant contains normal glycogen synthase and branching enzyme activity but has an ADPglucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and in contrast to the present strain enzyme studied previously, is highly active in the absence of the allosteric activator. The response of the CL1136 enzyme to energy charge has been determined and this enzyme shows appreciable activity at low energy charge values where the E. coli B enzyme is inactive. The response to energy charge for the CL1136 and E. coli B enzymes are correlated with the rates of glycogen accumulation observed in the microorganisms. The regulation of glycogen synthesis in E. coli is to a great extent at the level of ADPglucose pyrophosphorylase; varying concentrations of fructose-P₂ and energy charge determine the rate of ADPglucose and glycogen synthesis. Both the allosteric regulation of ADPglucose pyrophosphorylase as well as the genetic regulations of the synthesis of glycogen biosynthetic enzymes (glycogen synthase and ADPglucose pyrophosphorylase) are involved in the regulation of glycogen accumulation in E. coli B.     

Characterization of an autonomously activated plant ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize (Zea mays)/potato (Solanum tuberosum) small subunit mosaic, MP [Mos(1–198)], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme, was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here, we show that this mosaic, in the absence of activator, performs like a wild-type AGPase that is partially activated with 3-phosphoglyceric acid (3-PGA). In the presence of 3-PGA, enzyme properties of Mos(1–198)/SH2 are quite similar to those of the wild-type maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates, and partial inactivation by inorganic phosphate. The Mos(1–198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1–198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl-terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1–198)/SH2.     

Involvement of arginine residues in the allosteric activation and inhibition ofSynechocystis PCC 6803 ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacteriumSynechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl₂ enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form.V _max at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.     

Purification and preliminary characterization of sucrose-phosphate synthase using monoclonal antibodies

Monoclonal antibodies specific for sucrose phosphate synthase (SPS; EC 2.4.1.14) have been obtained for the first time. Three independent clones have been isolated which inhibited spinach (Spinacia oleracea L.) leaf SPS activity and facilitated the enzyme purification by immunoprecipitation. All three clones were specific for the spinach enzyme but neither inhibited nor precipitated the SPS present in tissue extracts of maize (Zea mays L.), barley (Hordeum vulgare L.), soybean (Glycine max L.), and sugar beet (Beta vulgaris L.). The inhibition of SPS activity by all three clones was reversible in the presence of UDPG, suggesting the presence of an epitope at the substrate-binding site. Immunoprecipitates of active enzyme preparations consistently revealed the presence of a 120 kilodalton polypeptide, indicating that the enzyme may be a homotetramer with a native molecular weight of about 480 kilodaltons. The occasional appearance of a 52 kilodalton polypeptide in the immunoprecipitates of some enzyme preparations was not the result of proteolysis, was not necessary for enzyme activity, and did not contain an antigenic site as revealed by Western blotting experiments. All three antibodies bind weakly to the SDS denatured 120 kilodalton subunit bound to nitrocellulose. The specific activity of the purified spinach enzyme was determined for the first time to be approximately 150 units per milligram SPS protein (pH 7.5 and 25°C) based on quantitative immunoprecipitation of the enzyme.     

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.     

Immunological comparison of the starch branching enzymes from potato tubers and maize kernels

Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M_r = 79,000) shares a high degree of similarity with maize branching enzyme I (M_r = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.     

Regulation of Starch Synthesis in the Bundle Sheath and Mesophyll of Zea mays L. : Intercellular Compartmentalization of Enzymes of Starch Metabolism and the Properties of the ADPglucose Pyrophosphorylases

The intercellular localization of enzymes involved in starch metabolism and the kinetic properties of ADPglucose pyrophosphorylase were studied in mesophyll protoplasts and bundle sheath strands separated by cellulase digestion of Zea mays L. leaves. Activities of starch synthase, branching enzyme, and ADPglucose pyrophosphorylase were higher in the bundle sheath, whereas the degradative enzymes, starch phosphorylase, and amylase were more evenly distributed and slightly higher in the mesophyll. ADPglucose pyrophosphorylase partially purified from the mesophyll and bundle sheath showed similar apparent affinities for Mg²⁺, ATP, and glucose-1-phosphate. The pH optimum of the bundle sheath enzyme (7.0-7.8) was lower than that of the mesophyll enzyme (7.8-8.2). The bundle sheath enzyme showed greater activation by 3-phosphoglycerate than did the mesophyll enzyme, and also showed somewhat higher apparent affinity for 3-phosphoglycerate and lower apparent affinity for the inhibitor, orthophosphate. The observed activities of starch metabolism pathway enzymes and the allosteric properties of the ADPglucose pyrophosphorylases appear to favor the synthesis of starch in the bundle sheath while restricting it in the mesophyll.     

A Proteinase from Germinating Barley : I. Purification and Some Physical Properties of a 30 kD Cysteine Endoproteinase from Green Malt

A proteinase was purified from germinated barley (green malt from Hordeum vulgare L. cv Morex) by acidic extraction, ammonium sulfate fractionation and successive chromatographies on CM-cellulose, hemoglobin sepharose, Sephadex G-75 and organomercurial agarose columns. The overall purification and final recovery were 290-fold and 7.5%, respectively. The purified enzyme was homogeneous on analytical gel electrophoresis, yielding a single protein associated with protease activity. An apparent molecular weight of about 20 kilodaltons was estimated for the native enzyme from gel filtration. SDS-gel electrophoresis revealed a single polypeptide of about 30 kilodaltons. The optimum pH for the hydrolysis of hemoglobin was around 3.8. The enzyme was strongly inhibited by leupeptin but was insensitive to phenylmethylsulfonyl fluoride, indicating that it was a cysteine proteinase. It hydrolyzed several large proteins from various origins. The ability of the enzyme to digest barley storage proteins in vitro was examined using SDS-gel electrophoresis. The hydrolysis patterns obtained showed that the enzyme rapidly hydrolyzed the large hordein polypeptides into relatively small fragments. The results of this study suggest that this 30 kilodalton enzyme is one of the predominant cysteine proteinases secreted into the starchy endosperm during barley germination and that it plays a major role in the mobilization of storage proteins.     

Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase

Pyridoxal-P has been shown to be an activator of the spinach leaf ADP-glucose pyrophosphorylase. It has a higher apparent affinity than the physiological activator 3-phosphoglycerate but only activates the enzyme activity 6-fold whereas 3-phosphoglycerate gives a 25-fold activation. Reductive phosphopyridoxylation of the spinach leaf enzyme results in enzyme having less dependence on the presence of activator for activity. Labeled pyridoxal-P is incorporated into both the 54- and 51-kilodalton subunits of the spinach leaf enzyme. The incorporation is inhibited by the presence of either 3-phosphoglycerate or the allosteric inhibitor, inorganic phosphate, thus suggesting that pyridoxal phosphate is covalently bound to the allosteric activator site. The pyridoxal phosphate is bound to an epsilon-amino group of a lysine residue. The phosphopyridoxylated enzyme is more resistant to phosphate inhibition than the unmodified form. The modified 51-kDa subunit has been digested with trypsin, and the peptide containing the labeled pyridoxal phosphate has been purified via high performance liquid chromatography and sequenced. Comparison of this sequence with the deduced amino acid sequence of a rice endosperm cDNA clone indicates that the putative allosteric site of the 51-kDa subunit is close to the carboxyl-terminal. This is in contrast to what had been demonstrated for the position of the activator site of the Escherichia coli ADP-glucose pyrophosphorylase which was shown to be close to the amino-terminal of the subunit.     

Pea chloroplast glutathione reductase: purification and characterization

Glutathione reductase (EC 1.6.4.2) was purified from intact pea (Pisum sativum) chloroplasts by a method which includes affinity chromatography on ADP-agarose. Fractions from the affinity column which had glutathione reductase activity consisted of polypeptides of 60 and 32 kilodaltons. Separation of the proteins by electrophoresis on native gels showed that glutathione reductase activity was associated with 60 kilodalton polypeptides and not with the 32 kilodalton polypeptides. Antibodies to spinach whole leaf glutathione reductase (60 kilodaltons) cross-react with the chloroplast 60 kilodalton glutathione reductase but not the 32 kilodalton polypeptides. In the absence of dithiothreitol the 60 kilodalton polypeptides showed a shift in apparent molecular weight on sodium dodecyl sulfate gels to 72 kilodaltons. Dithiothreitol did not alter the activity of the chloroplast enzyme. Chloroplast glutathione reductase is relatively insensitive to NADPH.     

Involvement of arginine residues in the allosteric activation and inhibition ofSynechocystis PCC 6803 ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacteriumSynechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl₂ enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form.V _max at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.