BINDING OF SUBSTRATES TO HUMAN DEOXYCYTIDINE KINASE STUDIED WITH LIGAND-DEPENDENT QUENCHING OF ENZYME INTRINSIC FLUORESCENCE

Deoxycytidine kinase is a key enzyme in the salvage pathway, and its activity is required for 5'-phosphorylation of several important antiviral and cytostatic nucleoside analogues. It has recently been purified completely from human sources. Steady-state and time-resolved fluorescence of human deoxycytidine kinase was used to study its interaction with the substrates dCyd, dAdo, dUrd, dTTP, and the feedback inhibitor dCTP. Enzyme fluorescence quenching by dCTP, dCyd, dTTP, and dAdo was bimodal, and the best fits of the quenching patterns were obtained using two modified Stern-Volmer equations with two sets of quenching constants (K(sv)) and accessibility values (f(a)) fitted independently for ''low'' and ''high'' concentration ranges of ligands. The transition between these occurred at about 20 muM dCTP, 50 muM dCyd, 30 muM dTTP, and 180 muM dAdo. Enzyme fluorescence showed unimodal quenching by dAdo and 30% reduced accessibility of the binding site in the presence of dCyd. dUrd quenching was also unimodal with K(sv) = 0.0047 +/- 0.0007 muM-1 and f(a) = 0.75 +/- 0.05, hence in the same range as for the ''high'' concentration range of dAdo in the absence of dCyd, where they are 0.0025 +/- 0.0003 muM-1 and 0.73 +/- 0.03, respectively. Fluorescence quenching was used to directly determine enzyme-ligand binding and revealed bimodal binding of dCTP, dCyd, dTTP, and dAdo and unimodal binding of dUrd, and of dAdo in the presence of 0.1 muM dCyd. Transition between these two modes of binding occurred at the concentrations described above. At the high concentration range of ligands, binding is characterized by 1-2 order of magnitude lower association constants than at the low concentration range, and severalfold higher binding capacities. Stoichiometry calculation gives about 1 molecule of ligand bound per 61-kDa dimer enzyme, except for dCTP, where it is equal to 0.6 +/- 0.2. The transition between the native and the unfolded form of the enzyme showed a midpoint of unfolding equal to 3.2 M guanidine hydrochloride, and after denaturation, the emission peak was shifted from 332 to 350 nm, the excitation peak remained unchanged at 285 nm, and the fluorescence quantum yield had decreased 2-fold. High-resolution photon counting gave fluorescence lifetimes ranging from tens of picoseconds to 10 ns with the best nonlinear least-squares fits corresponding to a sum of four discrete exponential terms. Static quenching of three excited states and dynamic quenching of one excited state were observed on interaction with dCyd. The fluorescence anisotropy decays are typical for this size class of enzymes and suggest that human deoxycytidine kinase has a relatively rigid structure. Interaction with 10 muM dCyd did not affect the motion of tryptophans and did not lead to aggregation of the enzyme subunits. Several models to explain the binding properties of human deoxycytidine kinase are described.