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 indisting uishable nonpathogenic species. The hexokinase (ATP:D-hexose 6-phosphotrans ferase, EC 2.7.1.1) isoenzyme patterns distinguish the pathogenic and nonpa thogenic species. Both species possess two hexokinases with very similar mo lecular mass and different isoelectric points. In order to understand the r ole of the two different isoenzymes from E. histolytica, we purified the re combinant hexokinases HXK1 and HXK2 and examined substrate spectrum and kin etic properties. The two enzymes displayed similar temperature and pH optim a, 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 wel l by HXK1, but at a much lower rate by HXK2. We attempted to expand the sub strate spectrum off. histolytica HXK1 by modifying its active site to becom e similar to the active site of the fructose phosphorylating yeast hexokina se PII. None of the nine mutants gained any fructokinase activity, but all of them retained at least some glucokinase and mannokinase activity. Mannok inase activity was decreased drastically by two single amino acid exchanges , both of which contributed significantly:ro 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. (C) 2000 Elsevier Science B.V. All rights reserved.