α-1,4-D-Glucan phosphorylase of gram-positive Corynebacterium callunae: isolation, biochemical properties and molecular shape of the enzyme from solution X-ray scattering

Abstract

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.

Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

“…Phosphorylase activity was measured in the direction of phosphorolysis at 30 mC by using a continuous, coupled enzyme assay described recently [22]. α--glucose 1-phosphate (Glc 1-P) was determined by a discontinuous enzymatic assay [22], or by HP anion chromatography on a Dionex HPLC system, model DX-120 (Dionex, Sunnyvale, CA, U.S.A.) with conductivity detection.…”

“…α--glucose 1-phosphate (Glc 1-P) was determined by a discontinuous enzymatic assay [22], or by HP anion chromatography on a Dionex HPLC system, model DX-120 (Dionex, Sunnyvale, CA, U.S.A.) with conductivity detection. PLP was measured by spectrophotometric (332 nm) or spectrofluorimetric (exc.…”

“…332 nm ; em. 550 nm) analyses [22]. Light scattering was measured on a Hitachi F2000 spectrofluorometer at 25 mC with excitation and emission wavelengths at 550 nm.…”

“…450 nm). Kinetic parameters of MalP and the Asn-133 Ala mutant were determined at 30 mC in phosphorolysis by using maltodextrin as the α-glucan substrate and measuring initial velocities with the discontinuous enzyme assay [22]. Parameter estimates were obtained from nonlinear fits of the data to a one-substrate Michaelis-Menten equation with substrate inhibition, using the least-squares method (Sigmaplot Vers.…”

“…Apart from the intrinsic protein absorbance at around 280 nm, the absorbance maximum of enzyme-bound PLP at 332 nm [22] and of free PLP at 405 nm can be used to monitor cofactor dissociation and denaturation. No significant changes in the absorbance spectrum of MalP occur between 25 and 40 mC.…”