Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae

Authors

  • Annick VANDERCAMMEN,

    1. Laboratoire de Chimie Physiologique, Université Catholique de Louvain and International Institute of Cellular and Molecular Pathology, Bruxelles
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  • Jean FRANÇOIS,

    1. Laboratoire de Chimie Physiologique, Université Catholique de Louvain and International Institute of Cellular and Molecular Pathology, Bruxelles
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  • Henri-Géry HERS

    Corresponding author
    1. Laboratoire de Chimie Physiologique, Université Catholique de Louvain and International Institute of Cellular and Molecular Pathology, Bruxelles
      Correspondence to H.-G. Hers, Laboratoire de Chimie Physiologique, UCL 7539, Avenue Hippocrate 75, B-1200 Bruxelles, Belgium
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Correspondence to H.-G. Hers, Laboratoire de Chimie Physiologique, UCL 7539, Avenue Hippocrate 75, B-1200 Bruxelles, Belgium

Abstract

The properties of yeast trehalose-6-phosphate synthase were reinvestigated in relation with the recent claim made by Panek et al. [Panek, A. C., de Araujo, P. S., Moura-Neto, V. and Panek, A. D. (1987) Curr. Genet. 11, 459–465] that the enzyme would be stimulated by ATP and partially inactivated by cAMP-dependent protein kinase. Trehalose-6-phosphate synthase activity was measured by the sum of [14C]trehalose 6-phosphate and [14C]trehalose formed from UDP-[14C]glucose and glucose 6-phosphate. The activity measured in an extract of Saccharomyces cerevisiae was not affected by any treatment of the cells, such as incubation in the presence of glucose or of dinitrophenol, which are known to greatly increase the intracellular concentration of cAMP, nor by preincubation of the extract in the presence of ATP-Mg, cAMP and bovine heart cAMP-dependent protein kinase. The activity was also not significantly different in several mutants affected in the cAMP system. The kinetic properties of the partially purified enzyme were investigated; no effect of ATP could be detected but Pi acted as a potent noncompetitive inhibitor (Ki= 2 mM). The activity of trehalose-6-phosphate phosphatae was measured by the amount of [14C]trehalose formed from [14C]trehalose 6-phosphate. The enzyme could be separated from other phosphatases and appeared to be highly specific for trehalose 6-phosphate. It was Mg dependent and its kinetics for trehalose 6-phosphate was hyperbolic. Studies performed with intact cells, crude extracts or the purified enzyme did not reveal any cAMP-dependent change in its activity.

Remarkably, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase copurified in the course of different chromatographic procedures, suggesting that they are part of a single bifunctional protein. A 50-fold purification of the two enzymes could be achieved with a yeild of only 2% by chromatography on Mono S followed by gel filtration on Superose 6B.

Abbreviations
Glc6P

glucose 6-phosphate

Tre6P

trehalose 6-phosphate

Fru(2,6)P2

2,6-bisphosphate

PFK-1

6-phosphofructo 1-kinase

Enzymes
 

Trehalose-6-phosphate synthase, α,α-trehalose-phosphate synthase (UDP-forming) (EC 2.4.1.15)

 

trehalose-6-phosphate phosphatase (EC 3.1.3.12)

 

trehalase (EC 3.2.1.28)

 

6-phosphofructo 1-kinase (EC 2.7.1.11)

 

glycogen synthase (EC 2.4.1.11)

Ancillary

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