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2002 | 49 | 3 | 651-658
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The effect of Arg209 to Lys mutation in mouse thymidylate synthase.

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Mouse thymidylate synthase R209K (a mutation corresponding to R218K in Lactobacillus casei), overexpressed in thymidylate synthase-deficient Escherichia coli strain, was poorly soluble and with only feeble enzyme activity. The mutated protein, incubated with FdUMP and N5,10-methylenetetrahydrofolate, did not form a complex stable under conditions of SDS/polyacrylamide gel electrophoresis. The reaction catalyzed by the R209K enzyme (studied in a crude extract), compared to that catalyzed by purified wild-type recombinant mouse thymidylate synthase, showed the Km value for dUMP 571-fold higher and Vmax value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios kcat, R209K/kcat, 'wild' and (kcat, R209K/Km, R209KdUMP)/( kcat, 'wild'/Km, 'wild'dUMP) were 0.019 and 0.000032, respectively, documenting that mouse thymidylate synthase R209, similar to the corresponding L. casei R218, is essential for both dUMP binding and enzyme reaction.
Physical description
  • >Nencki Institute of Experimental Biology, Warszawa, Poland
  • >Nencki Institute of Experimental Biology, Warszawa, Poland
  • Institute of Biology and Environmental Protection, Rzeszów University, Rzeszów, Poland
  • >Nencki Institute of Experimental Biology, Warszawa, Poland
  • Institute of Biotechnology and Antibiotics, Warszawa, Poland
  • >Nencki Institute of Experimental Biology, Warszawa, Poland
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