A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: A model for nucleoside kinase action

Deoxycytidine kinase (dCK) is an enzyme with broad substrate specificity wh ich can phosphorylate pyrimidine and purine deoxynucleosides, including imp ortant antiviral and cytostatic agents. In this study, stopped-flow experim ents were used to monitor intrinsic fluorescence changes induced upon bindi ng of various phosphate donors (ATP, UTP, and the nonhydrolyzable analogue AMP-PNP) and the acceptor dCyd to recombinant dCK. Monophasic kinetics were observed throughout. The nucleotides as well as dCyd bound to the enzyme b y a two-step mechanism, involving a rapid initial equilibrium step, followe d by a protein conformational change that is responsible for the fluorescen ce change. The bimolecular association rate constants for nucleotide bindin g [(4-10) x 10(3) M-1 s(-1)] were 2-3 orders of magnitude lower than those for dCyd binding [(1.3-1.5 x 10(6) M-1 s(-1)]. This difference most likely is due predominantly to the large difference in the forward rate constants of the conformational changes (0.04-0.26 s(-1) vs 560-710 s(-1)). Whereas t he kinetics of the binding of ATP, UTP, and AMP-PNP to dCK showed some diff erences, UTP exhibiting the tightest binding, no significant differences we re observed for the binding of dCyd to dCK in the presence or absence of ph osphate donors. However, the binding of dCyd to dCK in the presence of ATP or UTP was accompanied by a 1.5- or 3-fold higher quenching amplitude as co mpared with dCyd alone or in the presence of AMP-PNP. We conclude that ATP and UTP induce a conformational change in the enzyme, thereby enabling effi cient phosphoryl transfer.