KINETIC-ANALYSIS OF HUMAN DEOXYCYTIDINE KINASE WITH THE TRUE PHOSPHATE DONOR URIDINE TRIPHOSPHATE

Deoxycytidine kinase is the rate-limiting process in the activation fo r several clinically important antitumor agents. Previous studies have focused on deoxycytidine (dCyd) and adenosine triphosphate (ATP) as s ubstrates for this enzyme. In view of recent data indicating that urid ine triphosphate (UTP) is the physiologic phosphate donor for this enz yme, a study of the kinetic properties of dCyd kinase with dCyd and UT P was undertaken. The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phos phorylation by dCyd kinase. At high dCyd concentrations, dCyd kinase e xhibited substrate activation with ATP. In contrast, in the presence o f UTP, substrate inhibition was observed at concentrations of dCyd gre ater than 3 mu M. Inhibition by dCyd was noncompetitive with respect t o UTP and could not be reversed by a 200-fold increase in UTP concentr ation, indicating that the inhibition was not due to dCyd binding at t he nucleotide binding site. The kinetic mechanism for dCyd kinase was determined with dCyd and UTP as substrates. UTP was the preferred phos phate donor with a true K-m value of 1 mu M compared to 54 mu M with A TP, resulting in a 50-fold greater substrate efficiency for UTP. Altho ugh the double-reciprocal plots with UTP produced parallel lines, init ial velocity plots with other phosphate donors and product inhibition studies indicated that dCyd kinase formed a ternary complex with its s ubstrates. The parallel lines with UTP were apparently due to a low di ssociation constant for UTP, which was calculated as more than 13-fold lower than its K-m value. Analysis of product inhibition studies indi cated that dCyd kinase followed an ordered A-B random P-Q reaction seq uence, with UTP as the first substrate to bind. In contrast, previous results demonstrated a random bi-bi sequence for dCyd kinase in the pr esence of ATP. The combined results indicate that the enzyme can follo w a random bi-bi reaction sequence, but with UTP as the phosphate dono r, the addition of nucleotide prior to dCyd is strongly preferred. The noncompetitive substrate inhibition, which was independent of UTP con centration, indicates that high concentrations of dCyd promote additio n of the nucleoside prior to UTP, resulting in a lower velocity.