Characterization of Streptococcus pneumoniae thymidylate kinase: steady-state kinetics of the forward reaction and isothermal titration calorimetry.
Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis. The tmk gene from the bacterial pathogen Streptococcus pneumoniae was identified. The gene, encoding a 212-amino-acid polypeptide (23352 Da), was cloned and overexpressed in Escherichia coli with an N-terminal hexahistidine tag. The enzyme was purified to homogeneity, and characterized in the forward reaction. The pH profile of TMK indicates that its activity is optimal at pH 8.5. The substrate specificity of the enzyme was examined; it was found that not only ATP, but also dATP and to a lesser extent CTP, could act as phosphate donors, and dTMP and dUMP could serve as phosphate acceptors. Furthermore, AZT-MP (3'-azido-3'-deoxythymidine 5'-monophosphate) was shown not to be a substrate for S. pneumoniae TMK. Steady-state kinetics and inhibition studies with adenosine 5'-[beta-thio]diphosphate and dTDP in addition to isothermal titration calorimetry were performed. The data showed that binding follows an ordered pathway, in which ATP binds first with a K(m) of 235 +/- 46 microM and a K(d) of 116 +/- 3 microM, and dTMP binds secondly with a K(m) of 66 +/- 12 microM and a K(d) of 53 +/- 2 microM.
Petit
Chantal M
CM
Microbial Musculoskeletal and Proliferative Diseases, GlaxoSmithKline Pharmaceuticals, 1250 S. Collegeville Road, UP1345, Collegeville, PA 19426-0989, USA. Chantal_Petit-1@gsk.com
Koretke
Kristin K
KK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002151:Calorimetry; D003570:Cytidine Triphosphate / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D010766:Phosphorylation; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism
D003856:Deoxyuracil Nucleotides; D013942:Thymine Nucleotides; C019111:thymidine 3',5'-diphosphate; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; C007267:2'-deoxyuridylic acid; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase