Probing and Mapping the Pyridoxal 5′-Phosphate Site of Glycogen Phosphorylase

Shaltiel, Shmuel; Cortijo, Manuel; Zaidenzaig, Yeshayahu

doi:10.1007/978-3-662-37966-0_7

Cited by 2 publications

(9 citation statements)

References 59 publications

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“…However, absorption and fluorescence spectra of 7: thrrmophiliis phosphorylase reflect considerable differences in cofactor binding when compared with E. coli phosphorylase. As predicted from spectral analysis and model experiments with pyridoxal-P in organic solvents [25] and confirmed from structural studies [26] pyridoxal-P in phosphorylase is embedded in a hydrophobic microenvironment. It is likely that the microenvironment of the 7: thermophilus pyridoxal-P cofactor is more hydrophilic when compared with other phosphorylases.…”

Section: Discussionmentioning

confidence: 68%

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Purification and Characterisation of an α‐Glucan Phosphorylase from the Thermophilic Bacterium Thermus thermophilus

Boeck

Schinzel

1996

European Journal of Biochemistry

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An a-glucan phosphorylase has been purified 4500-fold from the thermophilic bacteria Thermus thermophilus. In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose. 7: tliernzophilus phosphorylase shares major properties with known mesophilic phosphorylases such as pyridoxal 5'-phosphate content (1 M pyridoxal-PIM subunit), subunit molecular mass (about 90 kDa) and inhibitor constants. The optimum temperature of 7: therniophilus phosphorylase was observed at 70°C in the pH range 5. 5-6.5. While at 25°C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70°C seems to be a dimer. Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other a-glucan phosphorylases. Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (V,,,;,, = 18 U/mg). Correspondingly, maltotriose functions ,as primer in the synthesis direction. Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenatc acting as a substrate indicate that the active site structure of 7: thermophilus phosphorylase differs from that of known rr-glucan phosphorylases.Keywnrds: glycogen phosphorylase ; thermophilic : Therrnus ; pyridoxal 5'-phosphate.Pyridoxal-5'-phosphate(pyridoxal-P)-dependent a-glucanphosphorylases catalyse the first step in the mobilisation of storage polysaccharides, resulting in the energy-conserving release of a-u-glucose 1 -phosphate (Glc-I 2'). All known a-glucanphosphorylases share the absolute requirement for pyridoxal-P linked to the c-amino group of a conserved lysine residue. Studies with pyridoxal-P derivatives, 31P-NMR and glycosylic substrate analogs r'evealed, that the close contact of the cofactor phosphate to the substrate phosphate is essential for activity. The non-hydrolytic cleavage in an aqueous environment requires complex interactions between cofactor, substrates and enzyme protein and the exclusion of water. In contrast to most other vitamin-B6-dependent enzymes, catalysis in phophorylases is dependent on the ionisable phosphate residue of the enzymebound pyridoxal-P (for a review see [I -31).Beside sharing pyridoxal-P as cofactor, a-glucan-phosphorylases from various sources have some features in common, such as their kinetic mechanism and their subunit molecular masses around 90 kDa. The amino acid sequence of domains involved i n substrate binding and catalysis is highly conserved (92 -100 %) among all known a-glucan-phosphorylases. Partial or complete sequeince divergence characterises those regions of phosphorylases that were believed to participate in individual responses to polysaccharide binding, allosteric effectors or protein phosphorylation. All known bacterial phosphorylases are devoid of allosteric or covalent regulation but subject to transcript...

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Section: Discussionmentioning

confidence: 68%

“…Native phosphorylases are characterised by their large Stokes shift with a fluorescence maximum at 535 nm when excited at 345 nm [25]. This fluorescence quenches at pH values where enzyme denaturation is observed.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterisation of an α‐Glucan Phosphorylase from the Thermophilic Bacterium Thermus thermophilus

Boeck

Schinzel

1996

European Journal of Biochemistry

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“…We have shown, for example, that dinitrophenylation of glycogen apophosphorylase b tags cysteines only [26,27] yet thiolysis does not proceed to completion [28,29]. In fact it was possible to preferentially unmask one or another of three labeled cysteine residues and thus to correlate the removal of the label with restoration of catalytic activity as measured after reconstitution with pyridoxal 5'-phosphate [28,29].…”

Section: Sharpening the Specificity Ofdinitrophenylationmentioning

confidence: 97%

“…One of the structural features common to all glycogen phosphorylases isolated so far is the presence of one molecule of tightly bound PLP in each of the enzyme protomers [36][37][38]. This cofactor is indispensable for the catalytic activity of the enzyme, and several lines of evidence raise the possibility that PLP might be either at the catalytic site or at a site involved in the transfer of a regulatory signal from or to the enzyme [28].…”

Section: Mapping Of Enzyme Active Sitesmentioning

confidence: 99%

“…Mapping the environment of PLP in glycogen phosphorylase was attempted by labeling the functional groups that become exposed upon removal of the cofactor from the enzyme [26][27][28]. Since it has been shown that PLP is embedded in a hydrophobic pocket [28,[39][40][41] and in view of the Iipophylic nature of the DNP ring, dinitrophenylation seemed a promising method for selective labeling of this site.…”

Section: Mapping Of Enzyme Active Sitesmentioning

confidence: 99%

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Dinitrophenylation and Thiolysis as a Tool in Protein Chemistry

Shaltiel

1974

Israel Journal of Chemistry

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The thiolytic cleavage of 2,4 dinitrophenyl derivatives of sulfhydryls, imidazole imino-nitrogens, and phenolic hydroxyls converts dinitrophenylation into a reversible method for masking or labeling these functional groups. The merits of this method are illustrated in the synthesis of peptides and polyamino acids (including a potent synthetic immunogen), in sharpening the specificity of dinitrophenylation, in masking "super reactive" nucleophiles in proteins while labeling "buried" functional groups, and in the probing and three-dimensional mapping of enzyme active sites. Analytical techniques for monitoring the rate and extent of the thiolysis step are described.Dinitrophenylation has played a major role in protein chemistry, as a key reaction used in the identification of N-terminal amino acids and thus in the elucidation of amino acid sequences of proteins [1]. Being an activated aryl halide, FDNB* reacts not only with the N-terminal amino groups but also with sulfhydryls, imino-groups of imidazoles, phenolic hydroxyls and e-amines [2][3][4]. Attempts have been made to achieve group specificity in dinitrophenylation by using less reactive reagents (e.g, dinitrobenzenesulfonic acid [5]) or by performing the reaction at a pH at which some of the functional groups are relatively much less reactive [6]. However, in practice it was found that the reactivity of certain functional groups in a protein is so much influenced by their microenvironment that it is often possible to find reaction conditions under which FDNB can be led quite selectively not only to one type of functional group but rather to a unique group within a specific site of the protein [7][8][9][10][11].A few years ago we showed [12] that DNP derivatives of sulfhydryls, imidazole irnino-nitrogens, and phenolic hydroxyIs (but not of Q and e amino groups) can be easily displaced by thiols, thus converting dinitrophenylation into a reversible tech-

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Probing and Mapping the Pyridoxal 5′-Phosphate Site of Glycogen Phosphorylase

Cited by 2 publications

References 59 publications

Purification and Characterisation of an α‐Glucan Phosphorylase from the Thermophilic Bacterium Thermus thermophilus

Purification and Characterisation of an α‐Glucan Phosphorylase from the Thermophilic Bacterium Thermus thermophilus

Dinitrophenylation and Thiolysis as a Tool in Protein Chemistry

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