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 which can phosphorylate pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. In this study, stopped-flow experiments were used to monitor intrinsic fluorescence changes induced upon binding 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 by a two-step mechanism, involving a rapid initial equilibrium step, followed by a protein conformational change that is responsible for the fluorescence change. The bimolecular association rate constants for nucleotide binding [(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 the kinetics of the binding of ATP, UTP, and AMP-PNP to dCK showed some differences, UTP exhibiting the tightest binding, no significant differences were observed for the binding of dCyd to dCK in the presence or absence of phosphate 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 compared 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 efficient phosphoryl transfer.
Turk
B
B
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala.
Awad
R
R
Usova
E V
EV
Björk
I
I
Eriksson
S
S
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003841:Deoxycytidine / Q000737:chemistry / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014544:Uridine Triphosphate / Q000737:chemistry / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D003841:Deoxycytidine; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D003842:Deoxycytidine Kinase; D014544:Uridine Triphosphate