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The effect of Arg209 to Lys mutation in mouse thymidylate synthase

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Ciesla J., Golos B., Walajtys-Rode E., Jagielska E., Plucienniczak A., Rode W.

Acta Biochimica Polonica

2002

tom 49

nr 3

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, R209K dUMP)/ (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.

Ograniczanie wyników

1 Acta Biochimica Polonica

1 Ciesla J.

1 Golos B.

1 Jagielska E.

1 Plucienniczak A.

1 Rode W.

1 Walajtys-Rode E.

1 2002

Wyniki wyszukiwania

The effect of Arg209 to Lys mutation in mouse thymidylate synthase