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Hexokinase PII from Saccharomyces cerevisiae is regulated by changes in the cytosolic Mg2+-free ATP concentration
Abstract:
Hexokinase PII is not inhibited by high Mg-ATP concentrations if the Mg2+-free ATP is kept at low levels (0.01 mM) in the assay mixture. Hexokinase PI activity is not affected either by Mg2+-free ATP nor by free Mg2+ in the assay mixture. Thus, hexokinase PI and PII activities appear not to be regulated by substrate inhibition as proposed previously [Kopetzki, E. & Entian, K. D. (1985) Eur. J. Biochem. 146, 657-662]. However, the level of Mg2+-free ATP in the hexokinase PII assay mixture strongly affects the enzyme activity by decreasing the Vmax and increasing the Km value for Mg-ATP from 0.15 mM to 5.0 mM. The physiological role of this inhibition, which has not been described previously, was investigated by determining the cytosolic ATP and Mg2+ concentrations in yeast cells grown under derepressing and repressing conditions. Derepression is accompanied by an important loss of Mg2+ from the cells, maintaining the ATP concentration constant. This produces an increase of Mg2+-free ATP in the cytosol from 0.01 mM to 0.1 mM. This free ATP concentration would lead to a maximal inhibition of hexokinase PII.
Insights
Yeast hexokinase PII is regulated by Mg2+-free ATP, not substrate inhibition. Increased free ATP in yeast cytosol during derepression significantly inhibits hexokinase PII activity.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Hexokinase PI and PII enzymes are crucial for glucose metabolism.
- Previous studies suggested substrate inhibition regulates hexokinase activity.
Purpose of the Study:
- To investigate the regulatory mechanisms of yeast hexokinase PI and PII.
- To determine the physiological relevance of Mg2+-free ATP in regulating hexokinase PII activity.
Main Methods:
- Enzyme kinetics assays for hexokinase PI and PII.
- Measurement of cytosolic ATP and Mg2+ concentrations in yeast cells under different growth conditions.
Main Results:
- Hexokinase PI activity is unaffected by Mg2+-free ATP or free Mg2+.
- Hexokinase PII is not inhibited by high Mg-ATP if Mg2+-free ATP is low.
- Elevated Mg2+-free ATP (0.01–0.1 mM) in yeast cytosol significantly inhibits hexokinase PII by reducing Vmax and increasing Km for Mg-ATP.
Conclusions:
- Hexokinase PI and PII are not regulated by substrate inhibition as previously proposed.
- Mg2+-free ATP is a key physiological regulator of hexokinase PII activity in yeast.
- Changes in cellular Mg2+ levels during derepression lead to increased free ATP, causing maximal hexokinase PII inhibition.