Reaction of phosphorylase-a with α-D-glucose 1-phosphate and maltodextrin acceptors to give products with degree of polymerization 6-89.

A series of linear glucan saccharides (GS) with defined quantity and degree of polymerization (DP) were synthesized from α-d-glucose 1-phosphate (α-d-Glc 1-P) by phosphorylase-a. The GS product fractions with average DP 11, 22, 38, 52, 60, 70, and 79 were measured by HPSEC-ELSD system. Then the same seven fractions were resolved into individual peaks with DP: 6-14, 10-32, 27-55, 37-67, 44-75, 49-83 and 53-89 by HPAEC-PAD system. Results showed that measurement of α-d-Glc 1-P amount consuming during GS synthesis by both systems enable calculation of reaction yield. The reaction yield for the 24h biosynthesis of the GS product was 25.3% (measured by HPSEC-ELSD) or 29.1% (measured by HPAEC-PAD). The HPSEC-ELSD and HPAEC-PAD systems were also successfully used for phosphorylase-a activity measurement in order to perform its kinetic characterization. This study established feasible systems for preparation of various sizes of the GS with defined DP and quantity as well as characterization of phosphorylase-a kinetics.

Increased sensitivity of glycogen synthesis to phosphorylase-a and impaired expression of the glycogen-targeting protein R6 in hepatocytes from insulin-resistant Zucker fa/fa rats.

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.

CoMFA and docking studies on glycogen phosphorylase a inhibitors as antidiabetic agents.

Glycogen phosphorylase (GP(a)) is a specific target for the design of inhibitors and may prevent glycogenolysis under high glucose conditions in type II diabetes. The carboxamides first reported by Hoover D. J. et al. (J. Med. Chem. 1998, 41, 2934-2938) are one of the major classes of GP(a) inhibitors other than glucose derivatives. The recent, X-ray crystallographic analyses (Oikonomakos et al. Biochim. Biophys. Acta 2003, 1647, 325-332) have revealed a distinct mechanism of action for these inhibitors, which bind at a new allosteric site away from the inhibitory and catalytic sites. To elucidate the essential structural and physicochemical requirements responsible for binding to the GP(a) enzyme and to develop predictive models, CoMFA and docking studies have been carried out on a series of indole-2-carboxamide derivates. The CoMFA model developed using pharmacophoric alignments and hydrogen-bonding fields demonstrated high predictive ability against the training (r2 = 0.98, q2 = 0.68) and the test set (r2pred = 0.85). Further the superimposition of PLS coefficient contour maps from CoMFA with the GP(a) active site (PDB: 1lwo) has shown a high level of compatibility.

Mutants of phosphorylase a altered in recognition by protein phosphatase-1.

To develop our knowledge of specificity determinants for protein phosphatase-1, mutants of phosphorylase b have been converted to phosphorylase a and examined for their efficacy as substrates for protein phosphatase-1. Mutants focused on the N-terminal primary sequence surrounding the phosphoserine (R16A, R16E, and I13G) and at a site that interacts with the phosphoserine in phosphorylase a, (R69K and R69E). The success achieved studying protein kinase substrate specificity with peptide substrates has not extended to protein phosphatases. Protein phosphatases are believed to recognize higher order structure in substrates in addition to the primary sequence surrounding the phosphoserine or threonine. Peptide studies with protein phosphatase-1 have revealed a preference for basic residues N-terminal to the phosphoserine. Arginine 16 in phosphorylase a may be a positive determinant. In this work, protein phosphatase-1 preferred the positive charge on arginine 16. R16A exhibited a similar K(m) but reduced V(max), and R16E had an increased K(m) and a decreased V(max) when compared to phosphorylase. I13G had a similar K(m) but an increased V(max). The R69 mutants were also dephosphorylated preferentially over phosphorylase a. The K(m) for R69K was unchanged but had a higher V(max). R69E exhibited the most changes, with a 4-fold increase in K(m) and a 10-fold increase in V(max). These results suggest that proper presentation of the phosphoserine can greatly affect the rate of dephosphorylation.

Shotgun identification of protein modifications from protein complexes and lens tissue.

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

The amino-terminal tail of glycogen phosphorylase is a switch for controlling phosphorylase conformation, activation, and response to ligands.

Glycogen phosphorylase is a muscle enzyme which metabolizes glycogen, producing glucose-1-phosphate, which can be used for the production of ATP. Phosphorylase activity is regulated by phosphorylation/dephosphorylation, and by the allosteric binding of numerous effectors. In this work, we have studied 10 site-directed mutants of glycogen phosphorylase (GP) in its amino-terminal regulatory region to characterize any changes that the mutations may have made on its structure or function. All of the GP mutants had normal levels of activity in the presence of the allosteric activator AMP. Some of the mutants were observed to have altered AMP-binding characteristics, however. R16A and R16E were activated at very low AMP concentration and crystallized at low temperature, like the phosphorylated form of GP, phosphorylase a, and unlike the dephospho-form, phosphorylase b. This indicates that even without phosphorylation, the structures of these mutants are more like phosphorylase a than phosphorylase b. These mutants were also very poorly phosphorylated in the presence of the inhibitor glucose, while phosphorylase b was phosphorylated normally with this inhibitor present. In contrast to R16A and R16E, four other mutants behaved like phosphorylase b after phosphorylation. R69E was only partially activated by phosphorylation, and I13G, R43E, and R43E/R69E were completely inactive after phosphorylation. We propose a model for the many functions of the amino terminus to explain the many varied effects of these mutations.

Structural basis of the synergistic inhibition of glycogen phosphorylase a by caffeine and a potential antidiabetic drug.

Caffeine, an allosteric inhibitor of glycogen phosphorylase a (GPa), has been shown to act synergistically with the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarboxylate (W1807). The structure of GPa complexed with caffeine and W1807 has been determined at 100K to 2.3 A resolution, and refined to a crystallographic R value of 0.210 (Rfree = 0.257). The complex structure provides a rationale to understand the structural basis of the synergistic inhibition between W1807 and caffeine. W1807 binds tightly at the allosteric site, and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface which transform GPa to the T'-like state conformation already observed with GPa-glucose-W1807 complex. A disordering of the N-terminal tail occurs, while the loop of polypeptide chain containing residues 192-196 and residues 43'-49', from the symmetry related subunit, shift to accommodate W1807. Caffeine binds at the purine inhibitor site by intercalating between the two aromatic rings of Phe285 and Tyr613 and stabilises the location of the 280s loop in the T state conformation.

Effects of microcystins on phosphorylase-a binding to phosphatase-2A: kinetic analysis by surface plasmon resonance biosensor.

Cyclic heptapeptide microcystins are a group of hepatoxicants which exert the cytotoxic effects by inhibiting the catalytic activities of phosphatase-2A (PP-2A) and phosphatase-1 (PP-1) and thus disrupt the normal signal transduction pathways. Microcystins interact with PP-2A and PP-1 by a two-step mechanism involving rapid binding and inactivation of protein phosphatase catalytic subunit, followed by a slower covalent interaction. It was proposed that inactivation of PP-2A/PP-1 catalytic activity by microcystins precedes covalent adduct formation. In this study, we used a biosensor based on surface plasmon resonance (SPR) to examine the effects of three microcystins, MCLR, MCRR and MCYR, on the binding between PP-2A and its substrate, phosphorylase-a (PL-a), during the first step of the interaction. The SPR biosensor provides real-time information on the association and dissociation kinetics of PL-a with immobilized PP-2A in the absence and presence of microcystins. It was found that the affinity of PL-a to microcystin-bound PP-2A was four times smaller compared to unbound PP-2A, due to 50% decreases in the association rates and two-fold increases in dissociation rates of PL-a binding to PP-2A. The results suggest that the rapid binding of microcystins to the PP-2A catalytic site leads to the formation of a noncovalent microcystin/PP-2A adduct. While the adduct formation fully inhibits the catalytic activity of PP-2A, it only results in partial inhibition of the substrate binding. The similar effects of the three microcystins on PP-2A suggest that the toxins bind to PP-2A at the same site and cause similar conformational changes. The present work also demonstrates the potential application of biosensor technology in environmental toxicological research.

Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive.

Continuous enzymatic assay for phosphorylase kinase in a monocascade enzyme system.

A turbidimetric method for continuous monitoring of the enzymatic reaction catalyzed by rabbit skeletal muscle phosphorylase kinase has been developed. The reaction mixture contained the substrates of glycogen phosphorylase a, i.e., glycogen and glucose 1-phosphate (or P(i)), in addition to the usual components of the kinase reaction. The kinetics of the cascade enzyme system were followed by the change in glycogen concentration over time, as measured by the absorbance of the reaction medium at 360 nm. The reliability of this turbidimetric method for measuring phosphorylase kinase activity was proven by comparison with a commonly used radiochemical assay. We present here a newly developed method for calculating the initial rate of phosphorylase kinase reaction in our conjugated system. We demonstrate that our procedure is applicable for investigating the hysteretic properties of phosphorylase kinase.

Purification and properties of glycogen phosphorylase from the fat body of larval Manduca sexta.

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.

Analysis of dissociation and unfolding of oligomeric proteins using a flat bed gel electrophoresis at high pressure.

A slab gel electrophoresis apparatus with the ability to operate over a pressure range of 10(-3) to 2 kbar is described. The system presented here is an improvement of a previous apparatus (A. A. Paladini, J. L. Silva, and G. Weber, Anal. Biochem. 161, 358-364, 1987). It consists of a flat bed gel, with a significantly enlarged buffer reservoir, which eliminates the requirement of high concentrations of running buffers, and at the same time allows shorter runs, leading to enhanced resolution and reproducibility. The application of the method to the dissociation of the tetramer glycogen phosphorylase a as a function of hydrostatic pressure is described. The flat geometry of the apparatus allows for the first time the analysis of the stability of oligomers and their constituent subunits to chemical denaturation by urea gradient electrophoresis gels at high pressure. Dimeric hexokinase shows a reversible cooperative unfolding transition with a midpoint at 3.8 M urea. In contrast, the monomers unfold at very low urea concentration (< 1.0 M). The observed differences in stability validates oligomerization as an important stabilizing element of the protein structure.

Physical heterogeneity of muscle glycogen phosphorylase revealed by hydrostatic pressure dissociation.

Four independent methods that employ fluorescence spectroscopy show that the tetramer of glycogen phosphorylase A (GPA) from rabbit muscle is reversibly dissociated into monomers by hydrostatic pressures under 2.5 kbar, if aggregation of the monomers is prevented by the addition of 8% glycerol. The free energy of association at 20 degrees C (-32 kcal mol-1) depends upon a large entropy increase (T delta S = +65 kcal mol-1) that counteracts an unfavorable enthalpy of association of +33 kcal mol-1. The association volumes calculated from the pressure dependence of the dissociation are nearly 4-fold smaller than those calculated from the shift in dissociation pressure with concentration. The dimer obtained by dilution of GPA at atmospheric pressure differs from the hypothesized dimer intermediate in the pressure dissociation by the much larger monomer affinity of the former. Like other tetramers, GPA shows hysteresis of the pressure profile upon decompression and conformational drift of the dissociated monomers. By use of the energy transfer method it is demonstrated that the relaxation time for half-dissociation (5 min) is over an order of magnitude shorter than that for subunit exchange (118 min). In all three tetramers studied, lactate dehydrogenase, glyceraldehyde phosphate dehydrogenase, and glycogen phosphorylase, the deterministic character of the dissociation equilibrium under pressure and the anomalous concentration dependence of the pressure dissociation demonstrate that these tetramers are heterogeneous populations with regard to their free energy and/or volumes of association.

Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP.

The crystal structures of activated R state glycogen phosphorylase a (GPa) and R and T state glycogen phosphorylase b (GPb) complexed with AMP have been solved at 2.9 A, 2.9 A and 2.2 A resolution, respectively. The structure of R state GPa is nearly identical to the structure of sulphate-activated R state GPb, except in the region of Ser14, where there is a covalently attached phosphate group in GPa and a non-covalently attached sulphate group in GPb. The contacts made by the N-terminal tail residues in R state GPa at the subunit interface of the functionally active dimer are similar to those observed previously for T state GPa. The quaternary and tertiary structural changes on the T to R transition allow these interactions to be relayed to the catalytic site in R state GPa. The transition from the T state GPb structure to the R state GPa structure results in a change in the N-terminal residues from a poorly ordered extended structure that makes intrasubunit contacts to an ordered coiled conformation that makes intersubunit contacts. The distance between Arg10, the first residue to be located from the N terminus, in R state GPa and T state GPb is 50 A. One of the important subunit-subunit interactions in the dimer molecule involves contacts between the helix alpha 2 and the cap' (residues 35' to 45' that form a loop between the 1st and 2nd alpha helices, alpha 1' and alpha 2' of the other subunit. The prime denotes residues from the other subunit). The interactions made by the N-terminal residues induce structural changes at the cap'/alpha 2 helix interface that lead to the creation of a high-affinity AMP site. The tertiary structural changes at the cap (shifts 1.2 to 2.1 A for residues 35 to 45) are partially compensated by the quaternary structural change so that the overall shifts in these residues after the combined tertiary and quaternary changes are between 0.5 and 1.3 A. AMP binds to R state GPb with at least 100-fold greater affinity and exhibits four additional hydrogen bonds, stronger ionic interactions and more extensive van der Waals' interactions with 116 A2 greater solvent accessible surface area buried compared with AMP bound to T state GPb.(ABSTRACT TRUNCATED AT 400 WORDS)