Fluorescence energy transfer studies of human deoxycytidine kinase: role of cysteine 185 in the conformational changes that occur upon substrate binding.
Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the Förster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.
Department of Experimental Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada.
Rajam S
RS
Mani
Elena V
EV
Usova
Carol E
CE
Cass
Staffan
S
Eriksson
D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003842:Deoxycytidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004735:Energy Transfer; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008301:Maleimides / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009705:Nucleosides / Q000378:metabolism / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry
D005456:Fluorescent Dyes; D008301:Maleimides; D009705:Nucleosides; D010710:Phosphates; D014364:Tryptophan; C003944:N-(3-pyrene)maleimide; D003842:Deoxycytidine Kinase; D003545:Cysteine; D000409:Alanine