Systematic exploration of active site mutations on human deoxycytidine kinase substrate specificity.
Human deoxycytidine kinase (dCK) is responsible for the phosphorylation of a number of clinically important nucleoside analogue prodrugs in addition to its natural substrates, 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine. To improve the low catalytic activity and tailor the substrate specificity of dCK, we have constructed libraries of mutant enzymes and tested them for thymidine kinase (tk) activity. Random mutagenesis was employed to probe for amino acid positions with an impact on substrate specificity throughout the entire enzyme structure, identifying positions Arg104 and Asp133 in the active site as key residues for substrate specificity. Kinetic analysis indicates that Arg104Gln/Asp133Gly creates a "generalist" kinase with broader specificity and elevated turnover for natural and prodrug substrates. In contrast, the substitutions of Arg104Met/Asp133Thr, obtained via site-saturation mutagenesis, yielded a mutant with reversed substrate specificity, elevating the specific constant for thymidine phosphorylation by over 1000-fold while eliminating activity for dC, dA, and dG under physiological conditions. The results illuminate the key contributions of these two amino acid positions to enzyme function by demonstrating their ability to moderate substrate specificity.
Iyidogan
Pinar
P
Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA.
Lutz
Stefan
S
D019943:Amino Acid Substitution; D001665:Binding Sites; D003842:Deoxycytidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000737:chemistry / Q000378:metabolism
D013937:Thymidine Kinase; D003842:Deoxycytidine Kinase