Abstract

We demonstrate that human 2′-deoxycytidine kinase (dCK) is a nonenantioselective enzyme because it phosphorylates β-d-2′-deoxycytidine (d-dCyd), the natural substrate, and β-l-2′-deoxycytidine (l-dCyd), its enantiomer, with the same efficiency. Kinetic studies showed thatl-dCyd is a competitive inhibitor of the phosphorylation ofd-dCyd with a K _ivalue of 0.12 μm, which is lower than theK _m value ford-dCyd (1.2 μm). Chemical modifications of either the base or the pentose ring strongly decrease the inhibitory potency of l-dCyd.l-dCyd is resistant to cytidine deaminase and competes in cell cultures with the natural d-dCyd as substrate for dCK, thus reducing the incorporation of exogenous [³H]dCyd into DNA. l-dCyd had no effect on the pool of dTTP deriving from the salvage or from the de novo synthesis, does not inhibit short term RNA and protein syntheses, and shows little or no cytotoxicity. Our results indicate a catalytic similarity between human dCK and herpetic thymidine kinases, enzymes that also lack stereospecificity. This functional analogy underlines the potential role of dCK as activator ofl-deoxycytidine analogs as antiviral and antineoplastic agents and lends support to the hypothesis that herpesvirus thymidine kinase might have evolved from a captured cellular dCK gene, developing the ability to phosphorylate thymidine and retaining that to phosphorylate deoxycytidine.