Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus.
Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30 degrees C. The optimum pH ranges for trehalose degradation and synthesis were 6.0-7.5 and 6.0-7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P(i) (K(i)=2.0 mM). The enzyme was highly specific towards trehalose, P(i), glucose and alpha-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P(i), glucose and alpha-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K(m) values were 61, 4.7, 24 and 6.3 mM for trehalose, P(i), glucose and alpha-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.
Wannet
W J
WJ
Department of Microbiology, Faculty of Science, University of Nijmegen, Netherlands.
Op den Camp
H J
HJ
Wisselink
H W
HW
van der Drift
C
C
Van Griensven
L J
LJ
Vogels
G D
GD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000364:Agaricus / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005947:Glucose / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature; D014199:Trehalose / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005958:Glucosephosphates; D008670:Metals; D000255:Adenosine Triphosphate; D014199:Trehalose; C031590:glucose-1-phosphate; D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase; D005947:Glucose