Hexokinase from Saccharomyces cerevisiae

Research area: Cell signalling. Hexokinase (HK) from yeast is a 486 amino-acid containing enzyme with a α/β fold. It comprises a large and small domain separated by an active site cleft. Conserved glycine residues are essential for ATP and glucose binding. The two isoforms are HKI and HKII encoded by HXK1 and HXK2 gene, respectively.

Hexokinase from Saccharomyces cerevisiae has been used:

• to investigate its amyloid fibril forming tendency using biophysical methods
• in tracking ATP depletion in peroxisomes and reticulocyte lysates in import assays
• in the synthesis of Mg-ADP-actin from Mg-ATP actin monomers

Catalyzes the phosphorylation of D-hexose sugars at the C6 position utilizing ATP as a phosphate source.

The rate of phosphorylation varies with different hexoses (pH 7.5, 30 °C).
D-fructose K_M: 0.33 mM
D-glucose K_M: 0.12 mM
D-mannose K_M: 0.05 mM

Yeast hexokinase exists as two similar isoforms, PI and PII (A and B), with isoelectric points of 5.25 and 4, respectively.

Molecular Weight: ~ 54 kDa (monomer)
~110 kDa (dimer)
Optimal pH: 7.5 to 9.0
Extinction Coefficient: E^1% = 8.85 (PI) and 9.47 (PII) at 280 nm

Activators: Hexokinase requires Mg²⁺ ions (K_M = 2.6 mM) for activity. Hexokinase is activated by catecholamines and related compounds.

Inhibitors: sorbose-1-phosphate, polyphosphates, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose, xylose, lyxose, and thiol reactive compounds (Hg²⁺ and 4-chloromercuribenzoate)

Hexokinase II is a crucial enzyme in glucose metabolism. Deletion of gene in S. cerevisiae in the presence of high glucose displays oxidative growth and results in the production of high biomass as well as shortened diauxic shift. Hexokinase II is also involved in glucose repression.

Lyophilized powder containing phosphate/citrate pH approx. 7.0

One unit will phosphorylate 1.0 μmole of D-glucose per min at pH 7.6 at 25 °C, unless otherwise indicated below.

Predominantly the PII isoform, produced from an overexpressing yeast strain.