Deoxycytidine kinase from calf thymus. Substrate and inhibitor specificity.

Kinetic constants were determined for 34 nucleoside substrates of deoxycytidine kinase (EC 2.7.1.74) from calf thymus. Substrate efficiency was assessed by the ratio of Vmax to Km. Inhibition constants were determined for 61 nonsubstrate nucleosides or nucleoside analogues. The enzyme was relatively specific for the pentose moiety of nucleoside substrates. beta-D-2'-Deoxyribonucleosides were more efficient substrates than the corresponding beta-D-arabinonucleosides. Unexpectedly, the L isomer of the beta-arabinonucleoside of cytosine was a more efficient substrate than was the D isomer. beta-Cytidine and beta-5-azacytidine were the only beta-D-ribonucleosides studied that had detectable substrate activity. alpha-Cytidine was an inhibitor but not a substrate. Nucleosides containing a variety of sugar moieties other than those mentioned above did not have detectable substrate activity. The enzyme was relatively nonspecific for the base moiety of nucleoside substrates. 2'-Deoxyribonucleosides of a variety of pyrimidines, purines, and other heterocycles were substrates. Cytosine was the most preferred pyrimidine moiety. 5-Substitution, except with fluorine, decreased substrate efficiency with nucleosides of cytosine or uracil. 2-Fluoradenine was the most preferred purine moiety. The effects of various purine ring substituents were interdependent. Nucleosides containing bulky, hydrophobic substituents on either the base or the pentose moiety had no substrate activity but were relatively potent competitive inhibitors. This suggested the presence of a hydrophobic region on the surface of the enzyme near the active site.