The polyphosphate- and ATP-dependent glucokinase from Propionibacterium shermanii: both activities are catalyzed by the same protein.
The glucokinase (EC 2.7.1.63) from Propionibacterium shermanii phosphorylates glucose using inorganic polyphosphate (poly(P)) or ATP as the phosphate donor. In this investigation, we have purified the glucokinase to homogeneity, using two methods and show that the polyphosphate and ATP-dependent glucokinase activities eluted as a single protein. The protein peak is shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse phase HPLC, and N-terminal sequence analysis. The purified protein eluted as a single peak from gel filtration and hydrophobic interaction HPLC columns and was found to display both the poly(P) and ATP glucokinase activities. Likewise, the two activities comigrated on a native isoelectric focusing gel. In addition, two analogues of ATP with different reactive groups displayed different inhibition patterns with respect to ATP and poly(P). The 2',3'-dialdehyde of ATP, whose reactive group is the dialdehyde of the ribose ring, showed competitive and noncompetitive patterns with respect to ATP and poly(P), respectively. While, 5'-p-fluorosulfonylbenzoyl adenosine, whose reactive sulfonyl fluoride group is related to the gamma-phosphoryl group of ATP, displayed competitive inhibition patterns with both ATP and poly(P). These observations provide evidence that the polyphosphate and ATP-dependent glucokinase activities of P. shermanii are the catalytic properties of a single enzyme and that the two substrates may have different binding sites on the enzyme with a common phosphorylating center.
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4983.
N F
NF
Phillips
P J
PJ
Horn
H G
HG
Wood
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005941:Glucokinase / Q000302:isolation & purification / Q000378:metabolism*; D006882:Hydroxyapatites; D007700:Kinetics; D011122:Polyphosphates / Q000378:metabolism*; D011424:Propionibacterium / Q000201:enzymology*; D013379:Substrate Specificity
D006882:Hydroxyapatites; D011122:Polyphosphates; D000255:Adenosine Triphosphate; D017886:Durapatite; D005941:Glucokinase