Differences in substrate specificity and kinetic properties of the recombinant hexokinases HXK1 and HXK2 from Entamoeba histolytica.
Entamoeba histolytica is responsible for amoebic colitis and liver abscess in humans. Entamoeba dispar is a closely related, morphologically indistinguishable nonpathogenic species. The hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns distinguish the pathogenic and nonpathogenic species. Both species possess two hexokinases with very similar molecular mass and different isoelectric points. In order to understand the role of the two different isoenzymes from E. histolytica, we purified the recombinant hexokinases HXK1 and HXK2 and examined substrate spectrum and kinetic properties. The two enzymes displayed similar temperature and pH optima, they were inhibited strongly by AMP and ADP, not by glucose 6-phosphate. Both enzymes phosphorylated glucose well and were unable to phosphorylate fructose or galactose. We also detected significant differences. HXK1 was more sensitive to inhibition by AMP and ADP. Mannose was phosphorylated well by HXK1, but at a much lower rate by HXK2. We attempted to expand the substrate spectrum of E. histolytica HXK1 by modifying its active site to become similar to the active site of the fructose phosphorylating yeast hexokinase PII. None of the nine mutants gained any fructokinase activity, but all of them retained at least some glucokinase and mannokinase activity. Mannokinase activity was decreased drastically by two single amino acid exchanges, both of which contributed significantly to this effect. The data indicate that a complex interaction of a number of amino acid residues is necessary for the ability to phosphorylate a given hexose.
Kroschewski
H
H
Institute for Specific Prophylaxis and Tropical Medicine, University of Vienna, Austria.
Ortner
S
S
Steipe
B
B
Scheiner
O
O
Wiedermann
G
G
Duchêne
M
M
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D006593:Hexokinase