Deoxynucleoside kinases encoded by the yaaG and yaaF genes of Bacillus subtilis. Substrate specificity and kinetic analysis of deoxyguanosine kinase with UTP as the preferred phosphate donor.
The overlapping yaaG and yaaF genes from Bacillus subtilis were cloned and overexpressed in Escherichia coli. Purification of the gene products showed that yaaG encoded a homodimeric deoxyguanosine kinase (dGK) and that yaaF encoded a homodimeric deoxynucleoside kinase capable of phosphorylating both deoxyadenosine and deoxycytidine. The latter was identical to a previously characterized dAdo/dCyd kinase (Møllgaard, H. (1980) J. Biol. Chem. 255, 8216-8220). The purified recombinant dGK was highly specific toward 6-oxopurine 2'-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2',3'-dideoxyguanosine. UTP was the preferred phosphate donor with a Km value of 6 microm compared with 36 microm for ATP. In addition, the Km for dGuo was 0.6 microm with UTP but 6.5 microm with ATP as phosphate donor. The combination of these two effects makes UTP over 50 times more efficient than ATP. Initial velocity and product inhibition studies indicated that the reaction with dGuo and UTP as substrates followed an Ordered Bi Bi reaction mechanism with UTP as the leading substrate and UDP the last product to leave. dGTP was a potent competitive inhibitor with respect to UTP. Above 30 microm of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.
Andersen
R B
RB
Department of Biological Chemistry, University of Copenhagen, Sølvgade 83, DK 1307 Copenhagen K, Denmark.
Neuhard
J
J
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005798:Genes, Bacterial; D007700:Kinetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; C019165:deoxyguanosine kinase