ATP-dependent glucokinase from the hyperthermophilic bacterium Thermotoga maritima represents an extremely thermophilic ROK glucokinase with high substrate specificity.
The gene (open reading frame (ORF) Tm1469, glk) encoding ATP-dependent ROK (repressors, ORFs, sugar kinases) glucokinase (ATP-GLK, EC 2.7.1.2) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 80 kDa composed of 36-kDa subunits. Rate dependence (at 80 degrees C) on glucose and ATP followed Michaelis-Menten kinetics with apparent Km values of 1.0 and 0.36 mM, respectively; apparent Vmax values were about 370 U mg(-1). The enzyme was highly specific for glucose as phosphoryl acceptor. Besides glucose only 2-deoxyglucose was phosphorylated to some extent, whereas mannose and fructose were not used. With a temperature optimum of 93 degrees C the enzyme is the most thermoactive bacterial ATP-GLK described.
Hansen
Thomas
T
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany. thansen@ifam.uni-kiel.de
Schönheit
Peter
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D003847:Deoxyglucose / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005632:Fructose / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005941:Glucokinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006358:Hot Temperature; D008358:Mannose / Q000378:metabolism; D008970:Molecular Weight; D021122:Protein Subunits / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics*
D021122:Protein Subunits; D011994:Recombinant Proteins; D005632:Fructose; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D005941:Glucokinase; D005947:Glucose; D008358:Mannose