Extending thymidine kinase activity to the catalytic repertoire of human deoxycytidine kinase.
Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K(m) values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.
Hazra
Saugata
S
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland (M/C 669), Chicago, Illinois 60607, USA.
Sabini
Elisabetta
E
Ort
Stephan
S
Konrad
Manfred
M
Lavie
Arnon
A
D000244:Adenosine Diphosphate; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D003561:Cytarabine / Q000378:metabolism; D003839:Deoxyadenosines / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013379:Substrate Specificity; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism*; D000093542:Gemcitabine
D003839:Deoxyadenosines; D050505:Mutant Proteins; D003561:Cytarabine; D003841:Deoxycytidine; D000244:Adenosine Diphosphate; D013937:Thymidine Kinase; C092717:thymidine kinase 1; D003842:Deoxycytidine Kinase; D003849:Deoxyguanosine; D013936:Thymidine; D000093542:Gemcitabine