Distinct sites for deoxyguanosine and deoxyadenosine phosphorylation on a monomeric kinase from Lactobacillus acidophilus.
Base-line separation of two paired deoxynucleoside kinase activities (deoxycytidine/deoxyadenosine and deoxyguanosine/deoxyadenosine kinase), previously resolved as overlapping peaks from Blue Sepharose, has now been achieved. The improved separation and recovery in relatively small volumes were accomplished by eluting Blue Sepharose with a bisubstrate mixture: 0.5 mM dCyd plus 1 mM ATP released dCyd/dAdo kinase, and 1 mM dGuo plus 5 mM ATP eluted dGuo/dAdo kinase. The latter pair of activities showed copurification through UDP-Sepharose affinity chromatography and HPLC anion-exchange chromatography. The HPLC preparation appeared to be homogeneous, on the basis of nondenaturing polyacrylamide gel electrophoresis at several gel concentrations and pH values. Both dGuo and dAdo kinase activities coincided with the protein band. A single band of protein was also observed upon sodium dodecyl sulfate gel electrophoresis. The estimated molecular weight of the denatured protein (56 000) agrees closely with values obtained for native activity by sedimentation equilibrium or gel permeation chromatography. The rate of dAdo phosphorylation was found to be stimulated more than 3-fold by the presence of dGuo, and dGuo kinase was also slightly activated by the presence of dAdo. This mutual activation indicates that dGuo and dAdo kinase activities do not share a common site. Selective chemical inactivation of dGuo kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine eliminated the ability of dGuo to stimulate dAdo kinase in parallel with the loss of dGuo kinase activity. These lines of evidence strongly suggest that dGuo and dAdo kinase activities are functions of separate sites on a monomeric polypeptide and that these sites may be in allosteric communication.
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels; D001665:Binding Sites; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003839:Deoxyadenosines / Q000378:metabolism*; D003842:Deoxycytidine Kinase / Q000378:metabolism; D003849:Deoxyguanosine / Q000378:metabolism*; D007700:Kinetics; D007779:Lactobacillus acidophilus / Q000201:enzymology*; D008970:Molecular Weight; D010766:Phosphorylation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011489:Protein Denaturation
D000345:Affinity Labels; D003839:Deoxyadenosines; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; C019165:deoxyguanosine kinase; D003842:Deoxycytidine Kinase; C020005:deoxyadenosine kinase; D003849:Deoxyguanosine; D000241:Adenosine