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.     

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.     

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 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.     

Isolation and Characterization of a Starchless Mutant of Arabidopsis thaliana (L.) Heynh Lacking ADPglucose Pyrophosphorylase Activity

A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F₁ progeny of a cross between the mutant and wild type had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus.     

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.     

Regulation of ADPglucose pyrophosphorylase in cucumber plants infected with the cucumber mosaic virus

ADPglucose pyrophosphorylase level and mechanisms regulating its activity were studied in cucumber plants infected with the cucumber mosaic virus at the stage of chronic infection. Studies carried out with partially purified preparations of the enzyme have shown that there was no substantial difference in the regulatory influence of the ratio 3-PGA/P₁, or in the number of binding sites of the effectors on the enzyme, but that the virus infection reduced the level of the enzyme in the tissues to 74% of the control and the 3-PGA/P₁ ratio to one half which resulted in a further decrease in ADPglucose pyrophosphorylase activity. In crude homogenate prepared from diseased plants, activity of the enzyme was reduced to 42% of the healthy control. The level of UDPglucose pyrophosphorylase was three times higher in cucumber leaf tissues than the level of ADPglucose pyrophosphorylase which was inhibited by both 3-PGA and P₁. Inhibitory effects of both these effectors were cumulated. The enzyme isolated from healthy plants was inhibited by inorganic phosphate more strongly than the enzyme isolated from diseased plants. UDPglucose pyrophosphorylase activity was increased in crude homogenate of diseased plants to 127% of the healthy control when the level of the enzyme was the same in the tissues of both healthy and diseased plants which was presumably connected with the enhanced rate of sucrose catabolism.     

Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm

ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles α-glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30°C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.     

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.     

Starch Synthesis in Developing Potato Tubers

The activities of enzymes involved in starch metabolism were measured at intervals during tuberization and the early stages of tuber growth in Solanum tubersum grown in water culture under controlled environmental conditions. Starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase activities all increased during tuber development, the most pronounced increases occurring in the activities of ADP-glucose pyrophosphorylase and phosphorylase. The activity ratio ADPglucose pyrophosphorylase/phosphorylase was lowest in slow growing tubers and hightest in fast growing tubers. In addition, high sugar concentrations in fast growing tubers and low sugar concentrations in slow growing tubers suggested that enzyme levels might be influenced by sugar concentration. The activities of starch synthase, phosphorylase and ADPglucose pyrophosphorylase were increased 2–2.5 fold by the presence of 100 mM K⁺. It is concluded that the major enzyme changes occur as a consequence of tuber initiation and that starch accumulation is controlled, at least in part, by the activities of ADPglucose pyrophosphorylase and phosphorylase.     

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.     

Studies on the effect of temperature on the activity and stability of cyanobacterial ADP-glucose pyrophosphorylase

The effect of temperature on the activity and stability of ADPglucose pyrophosphorylase from Anabaena PCC 7120 was studied. Experimental optima temperatures were found around 37-40 degrees C or 42-45 degrees C, depending on the absence or the presence of allosteric effectors in the assay medium, respectively. In the range of temperature where the enzyme is stable, curved Arrhenius plots were obtained, indicating a transition temperature between 9 and 12 degrees C. Since these results were observed for both the forward and reverse reaction, with two different sets of substrates and two entirely different assay procedures, it seems unlikely that the effect can be on any component of the system other than the enzyme itself. Results suggest that cyanobacterial ADPglucose pyrophosphorylase undergoes conformational changes at different temperatures, rendering structures with different catalytic efficiencies. The different structures of the enzyme were visualized by emission fluorescence. ADPglucose pyrophosphorylase was irreversibly inactivated when exposed to temperatures above 40 degrees C. Inactivation was dependent on temperature and followed first order kinetics. The substrate, ATP, and the allosteric effectors, 3PGA and Pi, effectively protected the enzyme against thermal inactivation. Protection afforded by ATP was affected by MgCl2. These results suggest that the binding of the effectors to the enzyme resulted in conformational changes of the protein, rendering structures more stable to temperature treatments. Similar structures could be adopted by the enzyme in different environments, since the higher stability was observed in media containing either high ionic strength or high hydrophobicity.     

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.     

Spinach Leaf Intra and Extra Chloroplast Phosphorylase Activities during Growth

The amino terminal sequence of the spinach (Spinacia oleracea L. cv Bloomsdale Long Standing) leaf cytoplasmic phosphorylase was determined and shown to have little similarity to the known sequence of the potato tuber phosphorylase. The antigenic reaction of spinach chloroplast phosphorylase and rabbit muscle phosphorylase a to antiserum prepared against spinach leaf cytoplasmic phosphorylase was tested. Neither phosphorylase gave a positive reaction when tested by immunodiffusion or neutralization of enzyme activity. The two spinach phosphorylases were assayed throughout the growth of the plant. Activity of cytoplasmic phosphorylase increased 4- to 8-fold at 30 to 35 days from sowing. Enzyme protein levels, as measured by antibody neutralization, increased by a similar amount. There was no corresponding increase in chloroplast phosphorylase activity. The chloroplast phosphorylase varied in parallel with the chloroplast enzyme ADPglucose pyrophosphorylase. Starch levels were high during the earlier stages of growth and then fell to a constant low level just before the increase in cytoplasmic phosphorylase. The results are discussed with respect to the relationship and functions of the two phosphorylases.     

Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P₂), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P₂. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (K _a) of spinach leaf PFP for Fru-2,6-P₂ in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the K _a of spinach leaf PFP for Fru-2,6-P₂ was up to 2000-fold greater than that measured in the optimised assay and V _max decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P₂). When assayed under optimum conditions in the gluconeogenic direction, the K _a of spinach leaf PFP for Fru-1,6-P₂ was approximately 50 μM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased K _a up to 25-fold, and decreased V _max by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the K _a of PFP for Fru-2,6-P₂ to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P₂ levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the K _a of PFP for Fru-1,6-P₂ to an extent that activation of PFP by this compound is unlikely to be physiologically relevant. Received: 21 July 2000 / Accepted: 15 September 2000     

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.     

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.     

Stabilization of Escherichia coli penicillin G acylase against thermal inactivation by cross-linking with dextran dialdehyde polymers

The thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 by cross-linking with dextran dialdehyde molecules, at a molecular mass of 11 500, 37 700 and 71 000 Da, was studied. The thermal inactivation mechanisms of the native and modified PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active but temperature-sensitive transient states. The highest enhancement to the thermostability of PGA was obtained using dextran-71000-dialdehyde modification, as a␣nearly ninefold increase at temperatures above 50 °C. The modification of PGA by dextran-11500-dialdehyde resulted in a considerable reduction of the V _m and K _m parameters of the enzyme. However, other dextran dialdehyde derivatives used for modification did not cause a meaningful change in either V _m and K _m. Modification by dextran dialdehyde derivatives did not result in significant change to either the optimal temperature or the activation energy of PGA. All modified PGA preparations showed lower inactivation rate constants but higher half-lives for inactivation than those of the native PGA at all temperatures studied. As indicated by the half-life times and k _i values, dextran 71000-dialdehyde was found to be more effective at cross-linking in the thermo-stabilization of PGA than any other agent studied in this work. Received: 3 December 1996 / Received revision: 17 March 1997 / Accepted: 22 March 1997