The alpha-1,4-D-glucan phosphorylase from gram-positive Corynebacterium callunae has been isolated and characterized. The enzyme is inducible approx. 2-fold by maltose, but remarkably not repressed by D-glucose. The phosphorylase is a homodimer with a stoichiometric content of the cofactor pyridoxal 5'-phosphate per 88-kDa protein subunit. The specificity constants (kcat/Km, glucan) in the directions of glucan synthesis and degradation are used for the classification of the enzyme as the first bacterial starch phosphorylase. A preference for large over small substrates is determined by variations in the apparent binding constants rather than catalytic-centre activities. The contribution of substrate chain length to binding energy is explained assuming two glucan binding sites in C. callunae phosphorylase: an oligosaccharide binding site composed of five subsites and a high-affinity polysaccharide site separated from the active site. A structural model of the molecular shape of the phosphorylase was obtained from small-angle solution X-ray scattering measurements. A flat, slightly elongated, ellipsoidal model with the three axes related to each other as 1:(0.87-0.95):0.43 showed scattering equivalence with the enzyme molecule. The model of C. callunae phosphorylase differs from the structurally well-characterized rabbit-muscle phosphorylase in size and axial dimensions.
Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 59-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures~Ն50 8C!, enzyme0oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of K sulfate ϭ 4.5 mM and K phosphate Ϸ 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by~a! an increase by about 5% in the relative composition of the a-helical secondary structure,~b! reduced 1 H0 2 H exchange, and~c! protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 59-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylasẽ probably due to tightened intersubunit contacts!, phosphate, and sulfate, this indirectly~1! preserves a functional active site up to Ϸ50 8C, and~2! stabilizes the covalent protein cofactor linkage up to Ϸ70 8C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 8C of Ϸ25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a Ϸ20 8C higher value on the temperature scale.Keywords: a-glucan phosphorylase; denaturation mechanism; oxyanion ligand; phosphate stabilization Unlike a great number of a-d-glycoside hydrolases, a-1,4-dglucan phosphorylases catalyze the degradation of a-1,4-d-glucan molecules by using phosphate rather than water as an acceptor of the transferred glucosyl moiety, as shown in Equation 1 where N is the degree of polymerization of the a-1,4-d-glucan.Therefore, interactions between the enzyme and the substrate phosphate play an essential role in the catalytic cycle of phosphorylase and contribute to binding energy in the ground state and the transition state of the reaction~Johnson et al
The cDNA encoding trehalose phosphorylase, a family GT-4 glycosyltransferase from the fungus Schizophyllum commune, was isolated and expressed in Escherichia coli to yield functional recombinant protein in its full length of 737 amino acids. Unlike the natural phosphorylase that was previously obtained as a truncated 61 kDa monomer containing one tightly bound Mg2+, the intact enzyme produced in E. coli is a dimer and not associated with metal ions [Eis, Watkins, Prohaska and Nidetzky (2001) Biochem. J. 356, 757-767]. MS analysis of the slow spontaneous conversion of the full-length enzyme into a 61 kDa fragment that is fully active revealed that critical elements of catalysis and specificity of trehalose phosphorylase reside entirely in the C-terminal protein part. Intact and truncated phosphorylases thus show identical inhibition constants for the transition state analogue orthovanadate and alpha,alpha-trehalose (K(i) approximately 1 microM). Structure-based sequence comparison with retaining glycosyltransferases of fold family GT-B reveals a putative active centre of trehalose phosphorylase, and results of site-directed mutagenesis confirm the predicted crucial role of Asp379, His403, Arg507 and Lys512 in catalysis and also delineate a function of these residues in determining the large preference of the wild-type enzyme for the phosphorolysis compared with hydrolysis of alpha,alpha-trehalose. The pseudo-disaccharide validoxylamine A was identified as a strong inhibitor of trehalose phosphorylase (K(i)=1.7+/-0.2 microM) that displays 350-fold tighter binding to the enzyme-phosphate complex than the non-phosphorolysable substrate analogue alpha,alpha-thio-trehalose. Structural and electronic features of the inhibitor that may be responsible for high-affinity binding and their complementarity to an anticipated glucosyl oxocarbenium ion-like transition state are discussed.
Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic a1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5¢-phosphatecontaining subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structurefunction assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg fi Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45°C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mM of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.Keywords: interface; oxyanion; phosphate; stabilization; subunit dissociation.Glycogen phosphorylases (GPs) catalyse degradation of glycogen and structurally related reserve polysaccharides in the cytosol to provide energy via the branch point metabolite a-D-glucose-1-phosphate. All known GPs are functional homodimers composed of % 90-kDa subunits and require pyridoxal 5¢-phosphate (PLP) cofactor for activity [1][2][3][4][5][6][7]. Although a very low basal activity may be present in the holoenzyme protomer, quarternary interactions clearly determine physiological levels of phosphorylase activity and are a prerequisite for the regulatory properties of eukaryotic GPs [8][9][10]. Forces that stabilize the dimer structure of GP are therefore essential to optimal enzyme function under physiological boundary conditions. GPs are a/b proteins that display a twodomain fold in which the N-terminal domain and the C-terminal domain are separated by a catalytic site cleft. The structural elements that comprise the subunit-subunit interface are located in the N-terminal domain. The dimer contact regions of regulated and nonregulated GPs share structural similiarity overall, but differ on the molecular level [3][4][5][6][7...
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