Human deoxythymidine kinase II: substrate specificity and kinetic behavior of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia.
Cytoplasmic and mitochondrial deoxythymidine kinase isozymes derived from the blast cells of acute myelocytic leukemia differ in their substrate specificity and kinetic behavior. These enzymes require divalent cations for their activity. The data suggest that the major role of idvalent cations is to chelate with ATP; the complex thus formed serves as the phosphate donor for the reaction. The activity of various triphosphate nucleosides as a phosphate donor for cytoplasmic deoxythymidine kinase is as follows: ATP = dATP greater than ara-ATP greater than GTP greater than CTP greater than dGTP = dCTP greater than dUTP, whereas for mitochondrial deoxythymidine kinase, the order of activity is ATP greater than CTP greater than UTP = dATP greater than ara-ATP greater than dGTP = dCTP greater than dUTP. Neither IdUTP nor dTTP could serve as a phosphate donor in the reaction catalyzed by either isozyme. From the many pyrimidine analogues tested for their binding affinity to each of these isozymes, I-dUrd and Br-dUrd had high good affinity which was equivalent to that of deoxythymidine. 5-Allyl-dUrd, 5-ethyl-dUrd, and 5-propyl-dUrd were only weakly bound to each isozyme. 5-I-dCyd, 5-Br-dCyd, dCyd, and 5-vinyl-dUrd were tightly bound to mitochondrial deoxythymidine kinase but not to the cytoplasmic isozyme. dTTP and I-dUTP are potent inhibitors of the reaction catalyzed by both isozymes. In contrast, dCTP and ara-CTP are potent inhibitors only of the mitochondrial isozyme, but not of the cytoplasmic isozyme. ATP-MG2+ acts as a sigmoidal substrate of the cytoplasmic isozyme with a"Km" of 0.22 mM, and as a regular substrate of the mitochondrial isozyme with a Km of 0.1 mM. Deoxythymidine acts as a regular substrate for both cytoplasmic and mitochondrial isozyme with a Km of 2.6 and 5.2 muM, respectively. Initial velocity as well as product inhibition studies suggest that the cytoplasmic isozyme catalyzes the reaction via a "sequential" mechanism. In contrast, mitochondrial deoxythymidine kinase catalyzes the reaction via a "ping-pong" mechanism.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001853:Bone Marrow / Q000201:enzymology*; D001854:Bone Marrow Cells; D003593:Cytoplasm / Q000201:enzymology*; D003853:Deoxyribonucleosides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D011485:Protein Binding; D011741:Pyrimidine Nucleosides / Q000494:pharmacology; D012265:Ribonucleotides / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism*; D014529:Uridine / Q000031:analogs & derivatives / Q000494:pharmacology
D003853:Deoxyribonucleosides; D003854:Deoxyribonucleotides; D007527:Isoenzymes; D011741:Pyrimidine Nucleosides; D012265:Ribonucleotides; D000255:Adenosine Triphosphate; D013937:Thymidine Kinase; D008274:Magnesium; D013936:Thymidine; D014529:Uridine