Purification and Characterization of the Hexokinase fromSchistosoma mansoni, Expressed inEscherichia coli

• Published in: Protein expression and purification Vol. 8; no. 3; pp. 374 - 380
• Main Authors: Armstrong, Robert L., Wilson, John E., Shoemaker, Charles B.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.11.1996
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1996.0113

The hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) ofSchistosoma mansonihas been expressed inEscherichia colias a fusion protein including an N-terminal polyhistidine tag and enterokinase cleavage site. The enzyme was purified by metal chelate chromatography to >95% homogeneity, based on analysis by SDS–gel electrophoresis and isoelectric focusing. The absorbance at 280 nm was 0.54 for a 1 mg/ml solution (molar extinction coefficient 2.7 × 104cm2mol). The pIof theS. mansonihexokinase was 6.0–6.2, slightly more acidic than the rat Type I isozyme (pI6.35). TheS. mansonienzyme migrated as a single band of activity during nondenaturing cellulose acetate electrophoresis; the mobility was slightly greater than the rat Type I isozyme, consistent with the estimated pI.TheKmvalues for substrates glucose and ATP were 128 ± 10 and 927 ± 41 μM, respectively. In accord with a previous report, theS. mansonihexokinase exhibited moderate sensitivity to inhibition (competitive vs ATP) by the product, glucose 6-phosphate, with aKi≈ 150 μM; the product analog, 1,5-anhydroglucitol 6-phosphate, was somewhat less effective as an inhibitor, withKi≈ 500 μM. These kinetic properties were not altered by removal of the N-terminal fusion partner by enterokinase treatment. Immunological crossreactivity between the rat Type I isozyme and theS. mansonihexokinase was demonstrated by immunoblotting, but this was markedly dependent on the preparation of antiserum used. The activity of the enzyme is apparently highly dependent on maintenance of free sulfhydryl groups. Activity was maintained during storage in the presence of monothioglycerol; activity lost during storage in the absence of monothioglycerol could be partially restored by treatment with this reagent.