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J Preiss

Publications and source records attributed to J Preiss.

At least 37 records · Page 2Linked to original sources
PubMed1999

Heat stability of the potato tuber ADP-glucose pyrophosphorylase: role of Cys residue 12 in the small subunit.

Most of the ADP-glucose pyrophosphorylases from different sources are stable to a heat treatment. We found that in the potato (Solanum tuberosum L.) tuber enzyme, the intermolecular disulfide bridge located between Cys12 of the small subunits is responsible for the stability at 60 degrees C. When this unique disulfide bond is cleaved the enzyme is stable up to 40 degrees C. Mutation of Cys12 in the small subunit into either Ala or Ser yielded enzymes with stability similar to the reduced form of the wild type. Concurrently, the enzyme with a truncated small subunit on the N-terminal was stable only up to 40 degrees C. Thus, the N-terminal is important for the stability of the enzyme because of the presence of a disulfide bond.

Adenosine Diphosphate Glucose↗
PubMed1999

Site-directed mutagenesis evidence for arginine-384 residue at the active site of maize branching enzyme II.

Essential arginine residues are suggested to be located at the active sites of maize branching enzymes (BE) based on the evidence that two arginine residues are conserved in all BE from various species and that as little as one arginine residue is located at the active site of maize BE by phenylglyoxal (PGO) modification. To determine the exact location of the active-site arginine residue in BE, we employed peptide mapping and site-directed mutagenesis approaches. A single trypsin-digested, [14C]PGO-labeled peptide was purified from maize BEII by two rounds of HPLC separation, but we failed to obtain amino acid sequencing information. Site-directed mutagenesis was then used to create one mutant (arginine-384 to alanine-384), R384A. Immunoblotting result showed that BEII protein was expressed at a similar level in the wild type and the R384A mutant. However, BE activity in the R384A mutant was only 1.4% of the wild type. These results support the conclusion that the conserved arginine-384 residue is important in BEII catalysis.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

Arginine residue 384 at the catalytic center is important for branching enzyme II from maize endosperm.

Branching enzyme (BE) belongs to the amylolytic family which contains four highly conserved regions. These regions are proposed to play an important role in catalysis as they are thought to be necessary for catalysis and/or binding the substrate. Only one arginine residue was found to be conserved in a catalytic center at the same position in all known sequences of BEs from various species as well as in the alpha-amylase enzyme family. In mBEII, a conserved Arg residue 384 is in catalytic region 2. We have used site-directed mutagenesis of the Arg-384 residue in order to study its possible role in BE. Previous chemical modification studies (H. Cao and J. Preiss, 1996, J. Prot. Chem. 15, 291-304) suggest that it may play a role in substrate binding. Replacement of Arg-384 by Ala, Ser, Gln, and Glu in the active site caused almost total inactivation. However, a conservative mutation of the conserved Arg-384 by Lys resulted in some residual activity, approximately 5% of the wild-type enzyme. The kinetics of the purified mutant R384K enzyme were investigated and no large effect on the Km of the substrate amylose for BE was observed. Thus, these results suggest that conserved Arg residue 384 in mBEII plays an important role in the catalytic function of BEs but may not be directly involved in substrate binding.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

The N-terminal region is important for the allosteric activation and inhibition of the Escherichia coli ADP-glucose pyrophosphorylase.

The ADPglucose pyrophosphorylase (EC 2.7.7.27) from Escherichia coli is allosterically activated by fructose 1,6-bisphosphate and inhibited by AMP. Proteolysis of the enzyme with proteinase K causes loss of activity and generates two peptides, 21 and 28 kDa, from the 49.7-kDa subunit. The presence of ADPglucose, Mg2+, and fructose 1, 6-bisphosphate during the incubation with proteinase K protected the enzyme activity and prevented cleavage at sites Met181-Ala182 and Phe192-Val193. Proteolysis of the protected enzyme removed 10 to 13 amino acids from the N-terminal and 2 amino acids from the C-terminal. The resulting enzyme was almost independent of the need for fructose 1,6-bisphosphate for maximal activity and insensitive to inhibition by AMP. The apparent affinity for the substrates was similar to that of the fully-activated wild-type enzyme. These data suggest that amino acid residues in the N-terminal portion and possibly the C-terminal portion of ADPglucose pyrophosphorylase are part of the regulatory domain of the enzyme, critical for allosteric regulation of the enzyme.

Allosteric Regulation↗
PubMed1998

Mechanism of reductive activation of potato tuber ADP-glucose pyrophosphorylase.

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5, 5'-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose. The activation process could be reversed by oxidation with 5, 5'-dithiobis(2-nitrobenzoic acid). Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed.

Adenosine Triphosphate↗
PubMed1998

Cloning and expression of the glgC gene from Agrobacterium tumefaciens: purification and characterization of the ADPglucose synthetase.

The gene encoding ADPglucose synthetase (EC 2.7.7.27) from Agrobacterium tumefaciens was isolated and expressed in Escherichia coli. The recombinant protein was purified to electrophoretic homogeneity in steps including ion-exchange and hydrophobic chromatography. The same purification procedure was utilized to purify ADPglucose synthetase from A. tumefaciens cells. The enzymes from the two sources were purified and characterized and were found to have identical kinetic, regulatory, and structural properties. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only one polypeptide band of 50 kDa was detected. In immunoblotting following electrophoresis, the 50-kDa band reacted with antibodies raised against the Escherichia coli ADPglucose synthetase; there was no reaction with antibodies raised against the spinach enzyme. The immunoreactivity of the A. tumefaciens ADPglucose synthetase was confirmed in antibody neutralization assays. Using gel filtration, the native enzyme was shown to be a tetramer. Fructose 6-phosphate and pyruvate were the most effective activators of the enzyme; maximal activation was observed in the ADPglucose synthesis direction, in which the enzyme was activated about ninefold by fructose 6-phosphate and fivefold by pyruvate. Both activators increased the affinity of the enzyme for the substrates ATP and glucose 1-phosphate. Inorganic orthophospate, ADP, AMP, and pyridoxal phosphate behaved as inhibitors of the enzyme. The distinctive regulatory properties of the enzyme from A. tumefaciens are compared with those of two enterobacterial enzymes and discussed in the context of their deduced amino acid sequences.

Agrobacterium tumefaciens↗
PubMed1998

Glutamate-459 is important for Escherichia coli branching enzyme activity.

The branching enzyme belongs to the amylolytic family, a group of enzymes that cleave and/or transfer chains of glucan. The amylolytic enzymes are homologous and all contain four conserved regions, proposed to contain the active site. By primary structure analysis, a conserved position unique to branching enzymes has been identified. This residue, which is either Asp or Glu, depending on the species, is located immediately after the putative catalytic Glu-458 (Escherichia coli numbering). Branching enzymes differ from other amylolytic enzymes in having this acid pair, and we asked if this motif could be essential for branching enzyme action. We used site-directed mutagenesis of the Glu-459 residue in the E. coli branching enzyme in order to determine the significance of the conserved Asp/Glu in branching enzymes. A substitution of Glu-459 to Asp resulted in increased specific activity compared to wild-type, suggesting that the mutation had created a more efficient enzyme. Changing Glu-459 to Ala, Lys, or Gln lowered the specific activities and altered the preferred substrate from amylose to amylopectin.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

Site-directed mutagenesis of a regulatory site of Escherichia coli ADP-glucose pyrophosphorylase: the role of residue 336 in allosteric behavior.

Site-directed mutagenesis was used to probe the role of glycine residue 336 in the regulatory properties of Escherichia coli ADP-glucose pyrophosphorylase. This residue was previously found to be changed from glycine to aspartate in the gene of an Escherichia coli mutant strain. The mutant enzyme had altered kinetic properties, including higher activity in the absence of the activator fructose 1,6-bisphosphate (FBP), higher apparent affinity for FBP and substrates, and lower apparent affinity for the inhibitor AMP. The observed changes in activity were caused by this single mutation, because the aspartate mutant was prepared from the wild-type gene. The kinetic properties of the site-directed mutant are identical to those of the enzyme from the mutant strain. A series of mutants was prepared to explore the effects of charge, size, shape, and hydrophobicity of the amino acid at residue 336 on the enzyme regulatory properties. All of the mutants, except for the lysine and arginine enzymes, were expressed and purified for kinetic analysis. The glycine-336 residue is able to tolerate diverse substitutions without compromise of catalytic activity. A range of allosteric changes was observed, with the most dramatic effects seen with the highly active aspartate enzyme and the low-activity G336Q mutant, which exhibited lower apparent affinities for activator and substrates and higher apparent affinity for inhibitor. The altered allosteric properties of the G336D mutant enzyme were almost completely abolished by substitution of asparagine. Thus, the aspartate negative charge is essential for the altered binding of effectors.

Adenosine Monophosphate↗
PubMed1998

A kinetic study of site-directed mutants of Escherichia coli ADP-glucose pyrophosphorylase: the role of residue 295 in allosteric regulation.

The effects of amino acid substitutions at residue 295 on the regulatory properties of Escherichia coli ADP-glucose pyrophosphorylase were studied. In previous studies, this residue, altered from proline to serine (P295S) in the gene of a mutant strain of E. coli, resulted in a high-activity form of enzyme [higher activity in absence of activator fructose 1,6-bisphosphate (FBP), higher apparent affinity for FBP and substrates, and lower apparent affinity for the inhibitor, AMP]. The effects of size and charge on this site were explored by replacing Pro with Gly, Asp, Asn, Gln, or Glu. All mutant enzymes were expressed and purified for kinetic analysis. All mutant enzymes, to varying extents, were in more active form than the wild-type enzyme. Enzymes with a substituted negative charge (P295D, P295E) had the highest activity in the absence of FBP, while the P295G enzyme was most similar to the wild type. The P295D and P295E enzymes had the lowest apparent affinities for AMP; this effect was partially abolished by the neutral substitutions P295N and P295Q. Another mutation, G336D, had previously been found to produce an even higher activity enzyme form. In order to examine interactions between substitutions at the 295 and 336 positions, the double mutant P295D-G336D was constructed and characterized. The double mutant enzyme was more active in the absence of FBP, with a higher affinity for FBP and a lower apparent affinity for AMP than either single mutated enzyme. The significance of residue 295 in regulation is discussed.

Adenosine Monophosphate↗
PubMed1998

Functional analysis of conserved histidines in ADP-glucose pyrophosphorylase from Escherichia coli.

Two absolutely conserved histidines and a third highly conserved histidine are noted in 11 bacterial and plant ADP-glucose pyrophosphorylases. These histidines were individually mutagenized in the E. coli enzyme to glutamine in order to determine their function. Glutamine mutations at residues 143 and 156 produced functional enzymes in cell extracts with slightly lower than wild-type specific catalytic activities and with same heat stability characteristics of the wild-type enzyme. Substitution of residue 83 with glutamine however produced an enzyme having decreased thermal stability. Additional mutageneses at residue 83 with asparagine, arginine, or aspartate gave rise to enzymes having a progressively decreasing trend in thermal stability. These mutants are more susceptible to proteolysis than wild-type enzyme. Kinetic analysis of H83Q and H83N indicates that histidine 83 is not involved in the catalytic mechanism or in substrate binding but possibly in maintenance of the active catalytic structure.

Base Sequence↗
PubMed1998

High-level expression of branching enzyme II from maize endosperm in Escherichia coli.

The gene that encodes the mature branching enzyme II (BEII) protein from maize (Zea mays L.) endosperm was amplified by means of a polymerase chain reaction technique and inserted into a T7-based expression vector. Although this has been an efficient expression system of maize BEII in Escherichia coli, an example is presented in this report which allows a greater expression of mBEII protein from the bacterial system by changing only one codon. The key to the level of expression appears to be related to the conversion of the third thymine base in the 285 position codon of the mBEII cDNA to cytosine without altering the encoded mBEII protein product. The crude cell extracts of enzyme prepared from E.coli exhibited seven-fold higher expression of branching enzyme activity compared to expression of the native enzyme. The enzymes from wild-type and the silent mutation genes were purified. The proteins were indistinguishable kinetically and immunologically. Thus, we obtained a significantly improved expression of mBEII protein in the bacterial system.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis.

Incubation of maize branching enzyme, mBEI and mBEII, with 100 microM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants in E. coli showed a significant decrease of the activity and the mutant enzymes had Km values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

Mutagenesis of the glucose-1-phosphate-binding site of potato tuber ADP-glucose pyrophosphorylase

Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg2+ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, SK198RLK213R, was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.

Journal Article↗
PubMed1998

ADP-Glucose pyrophosphorylase from potato tubers. Site-directed mutagenesis studies of the regulatory sites.

Several lysines (Lys) were determined to be involved in the regulation of the ADP-glucose (Glc) pyrophosphorylase from spinach leaf and the cyanobacterium Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711; Y. Charng, A.A. Iglesias, J. Preiss [1994] J Biol Chem 269: 24107-24113). Site-directed mutagenesis was used to investigate the relative roles of the conserved Lys in the heterotetrameric enzyme from potato (Solanum tuberosum L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9- and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12- and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg2+. These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.

Amino Acid Sequence↗
PubMed1997

Construction of chimeric enzymes out of maize endosperm branching enzymes I and II: activity and properties.

Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE II, respectively) have quite different properties, and to elucidate the domain(s) that determines the differences, chimeric genes consisting of part mBE I and part mBE II were constructed. When expressed under the control of the T7 promoter in Escherichia coli, several of the chimeric enzymes were inactive. The only fully active chimeric enzyme was mBE II-I BspHI, in which the carboxyl-terminal part of mBE II was exchanged for that of mBE I at a BspHI restriction site and was purified to homogeneity and characterized. Another chimeric enzyme, mBE I-II HindIII, in which the amino-terminal end of mBE II was replaced with that of mBE I, had very little activity and was only partially characterized. The purified mBE II-I BspHI exhibited higher activity than wild-type mBE I and mBE II when assayed by the phosphorylase a stimulation assay. mBE II-I BspHI had substrate specificity (preference for amylose rather than amylopectin) and catalytic capacity similar to mBE I, despite the fact that only the carboxyl terminus was from mBE I, suggesting that the carboxyl terminus may be involved in determining substrate specificity and catalytic capacity. In chain transfer experiments, mBE II-I BspHI transferred more short chains (with a degree of polymerization of around 6) in a fashion similar to mBE II. In contrast, mBE I-II HindIII transferred more long chains (with a degree of polymerization of around 11-12), similar to mBE I, suggesting that the amino terminus of mBEs may play a role in the size of oligosaccharide chain transferred. This study challenges the notion that the catalytic centers for branching enzymes are exclusively located in the central portion of the enzyme; it suggests instead that the amino and carboxyl termini may also be involved in determining substrate preference, catalytic capacity, and chain length transfer.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1997

Arginine294 is essential for the inhibition of Anabaena PCC 7120 ADP-glucose pyrophosphorylase by phosphate.

Treatment of ADP-glucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacterium Anabaena PCC 7120 with phenylglyoxal in 50 mM Hepes, pH 8.0, at 25 degrees C resulted in a time- and concentration-dependent loss of enzyme activity. Phosphate, the inhibitor, protected the enzyme from inactivation most effectively, while 3-P-glycerate, fructose-1,6-P2, pyridoxal-P, and ATP plus magnesium were also good protectors. After incubation with 2 mM phenylglyoxal for 1 h, the modified enzyme had a 10-fold lower apparent affinity for phosphate in the absence of the activator, 3-P-glycerate, than that of the wild-type enzyme. This result has implicated the involvement of an arginine residue at the allosteric sites, most probably the inhibitor-binding site, of ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena PCC 7120. In order to identify the arginine residue, five arginine residues, which are conserved in all higher-plant and cyanobacterial enzymes but not in enteric bacterial enzymes, were individually converted to alanine by site-directed mutagenesis. The mutant enzymes, R66A, R105A, R294A, and R385A, were purified, and the properties of these mutants were compared with the wild-type enzyme. Substitution of arginine294 with alanine resulted in an enzyme with more than 100-fold or 40-fold lower affinity for the inhibitor, phosphate, in the absence or presence of 3-P-glycerate, respectively. This mutation had no or lesser impact on the kinetic constants for the substrates and the activator, 3-P-glycerate.

Adenosine Triphosphate↗
PubMed1997

Comparing the properties of Escherichia coli branching enzyme and maize branching enzyme.

Escherichia coli glycogen branching enzyme (GBE) and maize starch branching enzymes I (SBEI) and II (SBEII) were expressed in E. coli and purified. E. coli GBE branched amylose at a higher rate than did SBEII, but branched amylose at a lower rate than did SBEI. Similar to SBEI, GBE branched amylopectin at a lower rate than did SBEII. High-performance anion-exchange chromatography analysis of the branched products produced by BE revealed the minimum chain length (cl) required for branching. While GBE and SBEII showed the same minimum cl [degree of polymerization (dp) 12] required for branching, SBEI had a slightly higher minimum cl (dp 16) requirement for branching. The major differences between GBE and SBE are their specificities in terms of the size of chains transferred. In comparison with SBE, GBE had a much narrower size range of chains transferred and transferred mainly shorter chains. While SBEI and SBEII produced a large number of chains ranging from dp 6 to over dp 30, GBE predominantly transferred chains ranging from dp 5 to 16 and produced only a very small number of long chains with dp greater than 20. Although it has been reported that SBEI and SBEII preferentially transfer longer and shorter chains, respectively (1), this study further defines the differences between SBEI and SBEII in the size of chains transferred. SBEI predominantly transfers longer chains with dp greater than 10, while producing few shorter chains with dp 3 to 5. In contrast, SBEII preferentially transfers smaller chains with dp 3 to 9, with the most abundant chains being dp 6 and 7. The significance of minimum chain-length requirement by SBE is discussed in setting the invariant size of amylopectin cluster size (9 nm).

1,4-alpha-Glucan Branching Enzyme↗