Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase.
Human and mouse deoxycytidine kinase (dCK) (EC 2.7.1.74) were cloned and expressed in Escherichia coli. Michaelis-Menten kinetics were determined for the purified enzymes with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), 2-chloro-2'-deoxyadenosine (CdA), 2',3'-dideoxycytidine (ddCyd) and 9-beta-D-arabinofuranosylguanine (araG) as substrates and ATP and UTP as phosphate donors. Both human and mouse dCK showed highest affinity to dCyd with Km values of 0.05-0.2 microM. The anti-leukaemic compound CdA was the superior substrate of the nucleoside analogues tested. Both enzymes were able to efficiently utilize ATP and UTP as phosphate donors. However, the use of UTP instead of ATP as phosphate donor decreased Km values for all substrates investigated. The kinetic properties of mouse and human dCK differed in that the human enzyme showed higher affinity for the substrates dAdo, CdA, ddCyd and araG. The human enzyme also showed higher affinity for ATP and UTP. The ability to phosphorylate dCyd was, however, similar for both human and mouse dCK. At physiological concentration of the feedback inhibitor dCTP, mouse dCK showed lower activity than human dCK for all substrates investigated.
Johansson
M
M
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Karlsson
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003001:Cloning, Molecular; D003842:Deoxycytidine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D051379:Mice; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014544:Uridine Triphosphate / Q000378:metabolism
D000596:Amino Acids; D003845:Deoxycytosine Nucleotides; D011994:Recombinant Proteins; C024107:2'-deoxycytidine 5'-triphosphate; D000255:Adenosine Triphosphate; D003842:Deoxycytidine Kinase; D014544:Uridine Triphosphate