alpha-1,4-D-glucan phosphorylase of gram-positive Corynebacterium callunae: isolation, biochemical properties and molecular shape of the enzyme from solution X-ray scattering.
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.
Weinhäusel
A
A
Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur (BOKU), Muthgasse 18, A-1190 Vienna, Austria.
Griessler
R
R
Krebs
A
A
Zipper
P
P
Haltrich
D
D
Kulbe
K D
KD
Nidetzky
B
B
D000818:Animals; D003352:Corynebacterium / Q000201:enzymology*; D018360:Crystallography, X-Ray; D009682:Magnetic Resonance Spectroscopy; D006005:Phosphorylases / Q000737:chemistry* / Q000302:isolation & purification*; D011487:Protein Conformation; D011817:Rabbits
D006005:Phosphorylases