Systematic exploration of active site mutations on human deoxycytidine kinase substrate specificity.

ABSTRACT: 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

SUBMITTER: Iyidogan P 

PROVIDER: S-EPMC2736870 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature