Kinetic analysis of human deoxycytidine kinase with the true phosphate donor uridine triphosphate.
Deoxycytidine kinase is the rate-limiting process in the activation for several clinically important antitumor agents. Previous studies have focused on deoxycytidine (dCyd) and adenosine triphosphate (ATP) as substrates for this enzyme. In view of recent data indicating that uridine triphosphate (UTP) is the physiologic phosphate donor for this enzyme, a study of the kinetic properties of dCyd kinase with dCyd and UTP was undertaken. The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phosphorylation by dCyd kinase. At high dCyd concentrations, dCyd kinase exhibited substrate activation with ATP. In contrast, in the presence of UTP, substrate inhibition was observed at concentrations of dCyd greater than 3 microM. Inhibition by dCyd was noncompetitive with respect to UTP and could not be reversed by a 200-fold increase in UTP concentration, indicating that the inhibition was not due to dCyd binding at the nucleotide binding site. The kinetic mechanism for dCyd kinase was determined with dCyd and UTP as substrates. UTP was the preferred phosphate donor with a true Km value of 1 microM compared to 54 microM with ATP, resulting in a 50-fold greater substrate efficiency for UTP. Although the double-reciprocal plots with UTP produced parallel lines, initial velocity plots with other phosphate donors and product inhibition studies indicated that dCyd kinase formed a ternary complex with its substrates. The parallel lines with UTP were apparently due to a low dissociation constant for UTP, which was calculated as more than 13-fold lower than its Km value. Analysis of product inhibition studies indicated that dCyd kinase followed an ordered A-B random P-Q reaction sequence, with UTP as the first substrate to bind. In contrast, previous results demonstrated a random bi-bi sequence for dCyd kinase in the presence of ATP. The combined results indicate that the enzyme can follow a random bi-bi reaction sequence, but with UTP as the phosphate donor, the addition of nucleotide prior to dCyd is strongly preferred. The noncompetitive substrate inhibition, which was independent of UTP concentration, indicates that high concentrations of dCyd promote addition of the nucleoside prior to UTP, resulting in a lower velocity.
Hughes
T L
TL
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109, USA.
Hahn
T M
TM
Reynolds
K K
KK
Shewach
D S
DS
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003841:Deoxycytidine / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D014544:Uridine Triphosphate / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D010710:Phosphates; D003841:Deoxycytidine; D000255:Adenosine Triphosphate; D003842:Deoxycytidine Kinase; D014544:Uridine Triphosphate