Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human, and the Drosophila melanogaster, deoxyribonucleoside kinases.
In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP). With dCK and ATP1, products were shown to be 5'-phosphates. With dCK, donor properties of the analogues were dependent on the nature of the acceptor, as with natural 5'-triphosphate donors. With dCK (dCyd as acceptor), Km and Vmax for the two 2'(3')-deoxyadenosine-3'(2')-triphosphates are similar to those for ATP. With dGK, Km values are higher than for ATP, while Vmax values are comparable. Kinetic studies further demonstrated Michaelis-Menten (non-cooperative) or cooperative kinetics, dependent on the enzyme employed and the nature of the donor. The physiological significance, if any, of the foregoing remains to be elucidated. The overall results are, on the other hand, highly relevant to studies on the modes of interaction of nucleoside kinases with donors and acceptors; and, in particular, to interpretations of the recently reported crystal structures of dGK with bound ATP, of dNK with bound dCyd, and associated modeling studies.
Krawiec
Krzysztof
K
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Warsaw, Poland.
Kierdaszuk
Borys
B
Kalinichenko
Elena N
EN
Rubinova
Elena B
EB
Mikhailopulo
Igor A
IA
Eriksson
Staffan
S
Munch-Petersen
Birgitte
B
Shugar
David
D
D000818:Animals; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D003838:Deoxyadenine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster; D005663:Furans / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008968:Molecular Conformation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
C115030:2'-deoxyadenosine-3'-triphosphate; C115029:3'-deoxyadenosine-2'-triphosphate; D003838:Deoxyadenine Nucleotides; D005663:Furans; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases