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 (Ksv) and accessibility values (fa) fitted independently for "low" and "high" concentration ranges of ligands. The transition between these occurred at about 20 microM dCTP, 50 microM dCyd, 30 microM dTTP, and 180 microM 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 Ksv = 0.0047 +/- 0.0007 microM-1 and fa = 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 microM-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 microM dCyd. Transition between these two modes of binding occurred at the concentrations described above.(ABSTRACT TRUNCATED AT 250 WORDS)
Kierdaszuk
B
B
Department of Medical Biophysics, Karolinska Institute, Stockholm, Sweden.
Rigler
R
R
Eriksson
S
S
D002793:Cholic Acids / Q000737:chemistry; D003842:Deoxycytidine Kinase / Q000737:chemistry / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D003902:Detergents; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D008024:Ligands; D017510:Protein Folding; D013379:Substrate Specificity
D002793:Cholic Acids; D003854:Deoxyribonucleotides; D003902:Detergents; D008024:Ligands; D003842:Deoxycytidine Kinase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate