Purification and characterization of the hexokinase from Schistosoma mansoni, expressed in Escherichia coli.
The hexokinase (ATP;D-hexose 6-phosphotransferase, EC 2.7.1.1) of Schistosoma mansoni has been expressed in Escherichia coli as 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 x 10(4) cm2 mol). The pI of the S. mansoni hexokinase was 6.0-6.2, slightly more acidic than the rat Type I isozyme (pI 6.35). The S. mansoni enzyme 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. The Km values for substrates glucose and ATP were 128 +/- 10 and 927 +/- 41 microM, respectively. In accord with a previous report, the S. mansoni hexokinase exhibited moderate sensitivity to inhibition (competitive vs ATP) by the product, glucose 6-phosphate, with a Ki approximately 150 microM; the product analog, 1,5-anhydroglucitol 6-phosphate, was somewhat less effective as an inhibitor, with Ki approximately 500 microM. 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 the S. mansoni hexokinase 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.
Armstrong
R L
RL
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Wilson
J E
JE
Shoemaker
C B
CB
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005947:Glucose / Q000737:chemistry; D006593:Hexokinase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012550:Schistosoma mansoni / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D006593:Hexokinase; D005947:Glucose