The effect of Arg209 to Lys mutation in mouse thymidylate synthase.
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 N(5,10)-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 K(m) value for dUMP 571-fold higher and V(max) value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios k(cat, R209K)/k(cat, 'wild') and (k(cat, R209K)/K(m, R209K)(dUMP))/( k(cat, 'wild')/K(m, '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.
Cieśla
Joanna
J
Nencki Institute of Experimental Biology, Warszawa, Poland.
Gołos
Barbara
B
Wałajtys-Rode
Elzbieta
E
Jagielska
Elzbieta
E
Płucienniczak
Andrzej
A
Rode
Wojciech
W
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D002384:Catalysis; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D007939:Leukemia L1210 / Q000378:metabolism; D008239:Lysine / Q000235:genetics; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D003856:Deoxyuracil Nucleotides; D011994:Recombinant Proteins; D001120:Arginine; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase; D008239:Lysine