Structural comparison of phosphorylases from different sources

All of the -glucan phosphorylases so far purified from diverse origins have similar molecular and catalytic properties, whereas they differ in regulatory properties and glucan specificities. The activity of the rabbit muscle enzyme is regulated by phosphorylation-dephosphorylation and activated by AMP. On the other hand, the potato and Escherichia coli enzymes exist only in the active form, and are unaffected by the nucleotide. To elucidate the structural bases for these differences, we have determined the complete amino acid sequence of potato phosphorylase and compared it with those of the rabbit muscle and E. coli enzymes. The monomer of the potato enzyme contains 916 amino acids with a molecular weight of 103,916. About one-fourth of the amino-terminal threonine is blocked by an acetyl group. Sequence comparison among these enzymes reveals the presence of a characteristic 78-residue insertion in the middle of the polypeptide chain of the potato enzyme. Except for the large inserted portion, 51 and 40% of the amino acids in the potato enzyme are identical with the rabbit muscle and E. coli enzymes, respectively. The regions relevant to the regulation of the activity are completely different among the three enzymes, whereas those involved in the catalytic reaction are well conserved. The potato enzyme sequence is consistent with the tertiary structure of the rabbit muscle enzyme. The 78-residue insertion is located at the junction of the amino- and carboxyl-terminal domains on the molecular surface near the glycogen-storage site. This insertion could account for the substrate discrimination of the potato enzyme. The molecular evolution of phosphorylase is discussed based on the structural comparison among the three enzymes.     

Primary structure of sweet potato starch phosphorylase deduced from its cDNA sequence

Sweet potato (Ipomoea batatas) starch phosphorylase cDNA clones were isolated by screening an expression library prepared from the young root poly(A)⁺ RNA successively with an antiserum, a monoclonal antibody, and a specific oligonucleotide probe. One cDNA clone had 3292 nucleotide residues in which was contained an open reading frame coding for 955 amino acids. This sequence was compared with those of potato (916 residues plus 50-residue putative transit peptide) and rabbit muscle (841 residues) phosphorylases. The sweet potato phosphorylase has an overall structural feature highly homologous to that reported for potato phosphorylase, in conformity with the finding that they belong to the same class of plant phosphorylase. High divergencies of the two enzymes are found in the about 70 residue N-termini each including a putative transit peptide, and the midchain 78 residue insert typical of type I plant phosphorylase. We consider that the very high dissimilarity found in the midchain inserts is related to the difference in proteolytic lability of the two plant phosphorylases. Some structural features of the cDNA clone were also discussed.     

Engineered plant phosphorylase showing extraordinarily high affinity for various alpha-glucan molecules.

alpha-Glucan phosphorylases are characterized by considerable difference in substrate specificities, even though the primary structures are well conserved among the enzymes from microorganisms, plants, and animals. The higher plant phosphorylase isozyme designated as type L exhibits low affinity for a large, highly branched glucan (glycogen), presumably due to steric hindrance caused by a unique 78-residue insertion located beside the mouth of the active-site cleft, whereas another isozyme without the insertion (designated as type H) shows very high affinity for both linear and branched glucans. Using the recombinant type L isozyme from potato tuber as a starting framework and aiming at altering its substrate specificity, we have genetically engineered the 78-residue insertion and its flanking regions. Firstly, removal of the insertion and connection of the newly formed C- and N-terminals yielded a totally inactive enzyme, although the protein was produced in Escherichia coli cells in a soluble form. Secondly, a chimeric phosphorylase, in which the 78-residue insertion and its flanking regions are replaced by the corresponding region of the type H isozyme, has been shown to exhibit high affinity for branched glucans (Mori, H., Tanizawa, K., & Fukui, T., 1993, J. Biol. Chem. 268, 5574-5581), but when two and four unconserved residues in the N-terminal flanking region of the chimeric phosphorylase were mutated back to those of the type L isozyme, the resulting mutants showed significantly lowered affinity for substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of cyanogen bromide fragments of potato phosphorylase

Amino acid sequence analysis of the cyanogen bromide peptides of potato alpha-glucan phosphorylase was undertaken for comparison with rabbit muscle glycogen phosphorylase and for elucidation of the structural bases for the differences in the catalytic and regulatory properties between the animal and plant enzymes. The potato enzyme was carboxymethylated and cleaved with cyanogen bromide. The 17 distinct fragments produced were isolated by a combination of gel filtration, sulfopropyl ion exchange chromatography, and high performance liquid chromatography. The molecular weights of these fragments are distributed in a range of 300 to 30,000. Fragment CI has a blocked amino terminus, and has the same amino acid sequence as CII, which has been assigned as the amino-terminal fragment of potato phosphorylase. The blocking group was deduced to be an acetyl group from the results of fast atom bombardment mass spectrometry of an amino-terminal pentapeptide. This paper describes the sequence determination of all the cyanogen bromide fragments of potato phosphorylase. The complete structure is presented in the following paper (Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236).     

Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli

Higher plant tissues contain two alpha-glucan phosphorylase isozymes (EC 2.4.1.1), types L and H, localized in the plastid and the cytoplasm, respectively. We already isolated and sequenced a cDNA clone encoding the type L isozyme. Presently, a cDNA clone encoding the type H counterpart was isolated from a cDNA library of immature potato tuber by plaque hybridization, using two oligonucleotide probes synthesized based on the partial amino acid sequences of the type H isozyme. The message encodes a polypeptide of 838 amino acid residues. Sequence comparison of the two potato tuber phosphorylase isozymes revealed two major distinctions; the type L isozyme contains a 78-residue insertion in the middle of the polypeptide chain as well as a 50-residue amino-terminal extension. Except for these extra portions, the two isozyme sequences show an identity of 63%. The entire structural gene for the type H isozyme was inserted 3'-downstream of the strong T7 RNA polymerase promoter in the expression plasmid pET-3b. Escherichia coli BL21 (DE3) cells carrying this plasmid produced active phosphorylase upon induction with isopropyl-beta-D-thiogalactoside at 22 degrees C. The expression is entirely dependent on the temperature; the bacteria did not produce a detectable amount of the active enzyme at 37 degrees C. Addition of pyridoxine to the culture medium was effective for the enzyme production.     

Subcellular immuno-localization, amino acid composition and partial amino acid sequences of α-l,4-glucan phosphorylase of Gracilaria spp (Rhodophyta)

Antibodies have been raised against an α-l,4-glucan phosphorylase (EC 2. 4. 1. 1) purified from the red alga Gracilaria chilensis. Localization of α-l,4-glucan phosphorylase in thin sections of G. chilensis and G. tenuistipitata was performed using immuno-gold labelling and transmission electron microscopy. The enzyme was localized in the cytosol and around the cytosolic starch granules of the algal cells. The labelling was not associated with the chloroplast or the cell wall. Amino acid composition of the red algal phosphorylase was quite similar to that of potato tuber and rabbit muscle phosphorylases. Partial amino acid sequences showed 48, 54 and 65% homology with the rabbit, potato and Escherichia coli enzymes, respectively.     

Effects of carnosine and anserine on muscle and non-muscle phosphorylases

Rabbit muscle phosphorylases a and b are activated by carnosine, whereas potato and yeast phosphorylases are inhibited at the same concentration of dipeptide. Rabbit muscle phosphorylase a is activated by anserine whereas the b form enzyme and the potato and yeast enzymes are inhibited by the dipeptide. The dipeptides affect the Vmax values for the enzymes rather than the substrate Km values. Kinetic analysis suggested that, for rabbit muscle phosphorylase, both dipeptides compete for occupancy of the same binding site(s) on the enzyme.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli

The glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli. The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues. The glgP gene product, a polypeptide of Mr 87,000, was confirmed by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequence showed that homology between glgP of E. coli and rabbit glgP, human glgP, potato glgP, and E. coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively. Within this homologous region, the active site, glycogen storage site, and pyridoxal-5'-phosphate binding site are well conserved. The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene.     

Temperature-sensitive production of rabbit muscle glycogen phosphorylase in Escherichia coli

In order to understand how allosteric switches regulate both the catalytic activity and molecular interactions of glycogen phosphorylase, it is necessary to design and analyze variant proteins that test hypotheses about the structural details of the allosteric mechanism. Essential to such an investigation is the ability to obtain large amounts of variant proteins. We developed a system for obtaining milligram amounts (greater than 20 mg/l) of rabbit muscle phosphorylase from bacteria. Phosphorylase aggregates as inactive protein when a strong bacterial promoter is used under full inducing conditions and normal growth conditions. However, when the growth temperature of bacteria expressing phosphorylase is reduced to 22 degrees C we obtain active muscle phosphorylase. The degree to which the induced expression of phosphorylase protein is temperature sensitive depends on the strain of bacteria used. New assay and purification methods were developed to allow rapid purification of engineered phosphorylase proteins from bacterial cultures. The rabbit muscle phosphorylase obtained from the bacterial expression system is enzymatically identical to the enzyme purified from rabbit muscle. The expressed protein crystallizes in the same conditions used for growing crystals of protein from rabbit muscle and the crystal form is isomorphous. Rabbit muscle phosphorylase is one of the largest oligomeric mammalian enzymes successfully expressed in Escherichia coli. Our results indicate that optimization of a combination of growth and induction conditions will be important in the expression of other heterologous proteins in bacteria.     

The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase.

In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification. In Escherichia coli, the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties. We report the crystal structure of E. coli maltodextrin phosphorylase refined to 2.4 A resolution. The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme. The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved. However, the relative orientation of the two subunits in E. coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit-subunit interfaces. The sequence changes result in loss of each of the control sites present in rabbit muscle GP. As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP. The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase.     

Antigenic determinants of acylphosphatase from porcine skeletal muscle

Analysis of the quantitative precipitin reaction of acylphosphatase from porcine skeletal muscle with rabbit antiserum indicated the presence of at least two antigenic determinants on the porcine enzyme molecule. Immunological cross-reactivities of acylphosphatases from equine and rabbit skeletal muscles were examined. In double immunodiffusion with the antiserum, the precipitin lines of the porcine and equine enzymes completely fused, while the rabbit enzyme gave no precipitin line. The reaction between the 125I-labeled porcine enzyme and its antibody was inhibited to the same extent by the porcine and equine enzymes, but not by the rabbit enzyme. The three enzymes were similar in net charge and molecular weight on polyacrylamide gel electrophoreses. No conformational difference among the three enzymes was observed in their circular dichroism spectra. The amino acid composition of the rabbit enzyme differed from those of the porcine and equine enzymes in the contents of Glu, Gly, Lys, and Arg. Differences in the sequence of the rabbit enzyme from that of the porcine enzyme were investigated by comparison of the peptide maps of the tryptic peptides of the two enzymes. Four peptides of the rabbit enzyme were located at different positions from those of the porcine enzyme. Three of the four peptides from both enzymes were sequenced and all the tryptic peptides of both enzymes were characterized by amino acid analysis. The tryptic peptides of rabbit enzyme were tentatively aligned on the basis of their amino acid compositions and sequence homologies, compared with the corresponding peptides of the porcine enzyme. Among five amino acid residues of the porcine enzyme, Arg-4, Asp-28, Arg-31, Glu-56, and Ile-68, which are replaced in the rabbit enzyme, Arg-4 and Asp-28 are considered to be included in the antigenic determinants.     

Complementation of subunits from glycogen phosphorylases of frog and rabbit skeletal muscle and rabbit liver.

Activity can be induced in potentially active rabbit skeletal muscle phosphorylase monomers covalently bound to Sepharose by noncovalent interaction with soluble subunits carrying inactive pyridoxal 5'-phosphate analogs or even salicyladlehyde. These analogs are themselves incapable of reconstituting active holophorphorylase from apophosphorylase. Phosphorylases with one intrinsically inactive and one potentially active subunit have about one half of the activity of the native phosphorylase dimer. The usefulness of this technique for subunit complementation was demonstrated by forming hybrid phosphorylases with inactive Sepharose-bound rabbit skeletal muscle subunits containing pyridoxal 5'-phosphate monomethylester and soluble activatable frog muscle and rabbit liver phosphorylase monomers. The inactive Sepharose-bound subunit induced in each case activity in the soluble subunit. But whereas the inactive rabbit muscle phosphorylase subunit even transmitted its characteristic temperature dependence of the rate of the reaction to the frog muscle subunit, it could not propagate its control properties to the liver enzyme. Differences of hybrid phosphorylases are related to immunological and amino acid divergencies among the component enzymes.     

Isoform diversity of phosphorylase kinase alpha and beta subunits generated by alternative RNA splicing

We have sequenced rabbit cDNAs that encode one isoform of the alpha subunit and two isoforms of the beta subunit of phosphorylase kinase, in addition to the single isoform from fast skeletal muscle that has been characterized to date for each subunit. All these isoforms are generated by alternative RNA splicing. The alpha subunit sequence obtained from slow skeletal muscle (soleus) is characterized by an internal deletion of 59 amino acids. This deletion is predominant in mRNA from slow muscle, heart, and uterus and accounts for the smaller alpha subunit variant (alpha') characteristic of phosphorylase kinase purified from slow muscle and heart. The beta subunit mRNA can be differentially spliced at two sites. In all tissues (except skeletal muscle) that were analyzed, an internal segment encoding 28 amino acids of the muscle sequence is replaced by a homologous sequence of identical length, presumably through the use of mutually exclusive exons. In brain and some other tissues, the deduced N-terminal sequence of the beta subunit is also changed. This is achieved by an insertion into the mRNA sequence that interrupts the initial reading frame after 25 codons and starts a new reading frame, encoding a different N terminus of 18 amino acids. This modification probably affects the major regulatory phosphorylation site of the beta subunit.     

Structural and functional properties of Drosophila melanogaster phosphorylase: comparison with the rabbit skeletal muscle enzyme

Glycogen phosphorylase isolated from Drosophila melanogaster contains one pyridoxal 5'-phosphate per subunit; the coenzyme is in a hydrophobic environment. Fruit-fly phosphorylase a has lower KM for glucose-1-phosphate and is less sensitive to allosteric inhibitors than the b form of the enzyme. The amino acid composition of Drosophila phosphorylase differs from that of rabbit skeletal muscle phosphorylase. These two enzymes give distinct one dimensional peptide maps. The distribution of reactive SH-groups is markedly different in the insect and vertebrate phosphorylase. Fruit-fly phosphorylase a is dephosphorylated by either rabbit or Drosophila protein phosphatase-1 at a slower rate than rabbit muscle phosphorylase a.     

Photooxidation of alpha-glucan phosphorylases from rabbit muscle and potato tubers.

Photooxidation of alpha-glucan phosphorylases from rabbit muscle and potato tubers in the presence of rose bengal leads to a rapid loss of enzymatic activity which follows first-order kinetics. The process is pH dependent, being more rapid at higher pH. The inactivation is closely related to the destruction of histidine residues in the enzyme. It is suggested that histidine residues are largely responsible for the loss of enzymatic activity in the photooxidation. The inactivation of potato phosphorylase is retarded by substrates, whereas that of the muscle enzyme is not. The rate of photoinactivation of muscle phosphorylase b is increased with AMP, and decreased with ATP, ADP, IMP and glucose-6-P. This finding is considered to be closely related to the allosteric transition of phosphorylase.     

A second L-type isozyme of potato glucan phosphorylase: cloning,antisense inhibition and expression analysis

In potato tubers two starch phosphorylase isozymes, types L and H, have been described and are believed to be responsible for the complete starch breakdown in this tissue. Type L has been localized in amyloplasts, whereas type H is located within the cytosol. In order to investigate whether the same isozymes are also present in potato leaf tissue a cDNA expression library from potato leaves was screened using a monoclonal antibody recognizing both isozyme forms. Besides the already described tuber L-type isozyme a cDNA clone encoding a second L-type isozyme was isolated. The 3171 nucleotide long cDNA clone contains an uninterrupted open reading frame of 2922 nucleotides which encodes a polypeptide of 974 amino acids. Sequence comparison between both L-type isozymes on the amino acid level showed that the polypeptides are highly homologous to each other, reaching 81–84% identity over most parts of the polypeptide. However the regions containing the transit peptide (amino acids 1–81) and the insertion sequence (amino acids 463–570) are highly diverse, reaching identities of only 22.0% and 29.0% respectively.Northern analysis revealed that both forms are differentially expressed. The steady-state mRNA levels of the tuber L-type isozyme accumulates strongly in potato tubers and only weakly in leaf tissues, whereas the mRNA of the leaf L-type isozyme accumulates in both tissues to the same extent. Constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of starch phosphorylase L-type activity in leaf tissue, but had only sparse effects in potato tuber tissues. Determination of the leaf starch content revealed that antisense repression of the starch phosphorylase activity has no significant influence on starch accumulation in leaves of transgenic potato plants. This result indicated that different L-type genes are responsible for the starch phosphorylase activity in different tissues, but the function of the different enzymes remains unclear.     

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.