Purification and characterization of a novel ADP-dependent glucokinase from the hyperthermophilic archaeon Pyrococcus furiosus.
Pyrococcus furiosus uses a modified Embden-Meyerhof pathway during growth on poly- or disaccharides. Instead of the usual ATP-dependent glucokinase, this pathway involves a novel ADP-dependent (AMP-forming) glucokinase. The level of this enzyme and some other glycolytic enzymes appeared to be closely regulated by the substrate. Growth on cellobiose resulted in a high specific activity of 0.96 units mg-1, whereas on pyruvate a 10-fold lower activity was found. The ADP-dependent glucokinase was purified 1350-fold to homogeneity. The oxygen-stable enzyme had a molecular mass of 93 kDa and was composed of two identical subunits (47 kDa). The glucokinase was highly specific for ADP, which could not be replaced by ATP, phosphoenolpyruvate, GDP, PPi, or polyphosphate. D-Glucose could be replaced only by 2-deoxy-D-glucose, albeit with a low efficiency. The Km values for D-glucose and ADP were 0.73 and 0.033 mM, respectively. An optimum temperature of 105 degrees C and a half-life of 220 min at 100 degrees C are in agreement with the requirements of this hyperthermophilic organism. The properties of the glucokinase are compared to those of less thermoactive gluco/hexokinases.
Department of Microbiology, Wageningen Agricultural University, The Netherlands.
S W
SW
Kengen
J E
JE
Tuininga
F A
FA
de Bok
A J
AJ
Stams
W M
WM
de Vos
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001105:Archaea / Q000201:enzymology* / Q000254:growth & development; D001234:Aspergillus niger / Q000201:enzymology; D001419:Bacteria / Q000201:enzymology; D002474:Cell-Free System; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005941:Glucokinase / Q000302:isolation & purification* / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D000244:Adenosine Diphosphate; D017886:Durapatite; D005941:Glucokinase