Unusual nucleoside triphosphate donors for nucleoside kinases: 3'-deoxyadenosine-2'-triphosphate and 2'-deoxyadenosine-3'-triphosphate.
Two non-conventional analogues of ATP, 3'-deoxyadenosine-2'-triphosphate (3'-d-2'-ATP) and 2'-deoxyadenosine-3'-triphosphate (2'-d-3'-ATP), the syntheses of which are described, were examined as potential phosphate donors for the nucleoside kinases: 2'-deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2). The reactions were monitored by means of a mixture of [gamma-32P]ATP and cold analogue, and/or with the use of 3H-labelled acceptors and cold donor. With dCK, using equimolar mixtures of ATP with each analogue, and dC as acceptor, phosphate transfer from 3'-d-2'-ATP and 2'-d-3'-ATP amounted to 34% and 14%, respectively. With each analogue used alone (each at concentration of 100 microM), phosphate transfer from 3'-d-2'-ATP was 55% that from ATP, and from 2'-d-3'-ATP 16%. With human TK2, and equimolar mixtures of [gamma-32P]ATP with each of the analogues, and 1 microM dT as acceptor, there was no detectable transfer from either analogue. But, when each analogue was used alone, phosphate transfer attained 11% and 5%, respectively, that for ATP alone. With the low affinity form of human TK1, and dT as acceptor, only low phosphate transfer occurred with either analogue used alone. Both compounds exhibited Michaelis-Menten kinetics (with significantly lower Vmax than ATP), while ATP exhibited cooperative kinetics with all three kinases.
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Poland.
K
K
Krawiec
B
B
Kierdaszuk
E N
EN
Kalinichenko
I A
IA
Mikhailopulo
D
D
Shugar
D002384:Catalysis; D003838:Deoxyadenine Nucleotides / Q000737:chemistry*; D003842:Deoxycytidine Kinase / Q000737:chemistry*; D006801:Humans; D010766:Phosphorylation; D013937:Thymidine Kinase / Q000737:chemistry*
C115030:2'-deoxyadenosine-3'-triphosphate; C115029:3'-deoxyadenosine-2'-triphosphate; D003838:Deoxyadenine Nucleotides; D013937:Thymidine Kinase; D003842:Deoxycytidine Kinase