Characterization of guanosine kinase from Brevibacterium acetylicum ATCC 953.
Guanosine kinase (GKase) activity was identified in a cell-free extract of Brevibacterium acetylicum ATCC 953. We have purified the enzyme 4000-fold from the cell-free extract to apparent homogeneity. Molecular weight of 71,300 and 36,300 determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively, revealed that the enzyme is a dimer molecule. Maximum activity of the GKase was attained when the magnesium/ATP concentration ratio was close to 1. The GKase activity was dependent on the presence of a divalent cation. The Km values for guanosine, inosine, and 2'-deoxyguanosine as phosphate acceptors were determined to be 0.022, 0.87, and 2.83 mM, respectively. In addition to ATP and dATP, GTP and dGTP were shown to be effective phosphate donors for the GKase. The optimal pH seemed to be around pH 8.3, although relatively high activity was observed in the alkaline pH range of 7.5-9.8. The addition of 0.1 mM pyrimidine nucleotides, especially CMP and CTP, activated the GKase activity. On the other hand, the addition of 1 mM AMP, ADP, and GMP inhibited the GKase activity. It is thus likely that the GKase activity might be regulated in vivo by nucleotide concentrations and may control the nucleoside monophosphate level by efficiently recycling guanosine.
Usuda
Y
Y
Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki-shi, Japan. lt_usuda@te10.ajinomoto.co.jp
Kawasaki
H
H
Shimaoka
M
M
Utagawa
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D001951:Brevibacterium / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D009711:Nucleotides; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C055032:guanosine kinase