Open AccessCloning and biochemical characterisation of Aspergillus niger hexokinase
Author(s) -
Panneman Henk,
Ruijter George J. G.,
van den Broeck Hetty C.,
Visser Jaap
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2580223.x
Subject(s) - hexokinase , glucokinase , biochemistry , fructose , enzyme , aspergillus niger , mannose , biology , fructokinase , trehalose , chemistry , glycolysis
The Aspergillus niger hexokinase gene hxk A has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe. The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts. The encoded protein was purified from a multicopy hxk A transformant, and extensively characterised. The hexokinase protein has a molecular mass of 54 090, a p I of 4.9 and is a homodimer. D ‐Glucose, the glucose analogue 2‐deoxy‐ D ‐glucose, D ‐fructose, D ‐mannose and D ‐glucosamine are phosphorylated by hexokinase, whereas the hexoses D ‐galactose, L ‐sorbose, methyl α‐ D ‐glucoside and the pentoses L ‐arabinose and D ‐xylose are not. The enzyme has high affinity for glucose ( K m = 0.35 mM at pH 7.5) and for fructose ( K m = 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1−0.2 mM) of trehalose 6‐phosphate, which may be of importance for in vivo regulation of the enzyme. Inhibition of A. niger hexokinase by trehalose 6‐phosphate is competitive towards the sugar substrate ( K i = 0.01 mM). Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration. At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM. Expression of the hexokinase and the glucokinase gene requires active carbon metabolism. Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed. The hexokinase and glucokinase enzymes are quite stable in vivo . Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3−4 h after depletion of the carbon source.