Trehalose-6-phosphate synthase/phosphatase complex from bakers' yeast: purification of a proteolytically activated form.
A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers' yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4.5-fold), and increased TPS activity at 5 mM-UDP-glucose/10 mM-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (Ki about 5 mM), but increased TPP activity about six-fold (Ka about 4 mM). Phosphate (6 mM) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.
Londesborough
J
J
Research Laboratories, Alko Ltd, Helsinki, Finland.
Vuorio
O
O
D004789:Enzyme Activation; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014199:Trehalose / Q000378:metabolism
D005958:Glucosephosphates; D009097:Multienzyme Complexes; D010710:Phosphates; D019298:Glucose-6-Phosphate; D014199:Trehalose; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase; C059055:N-acetylglucosaminyldiphosphoundecaprenol glucosyltransferase; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases