Aspartate 221 of thymidylate synthase is involved in folate cofactor binding and in catalysis.
Structural studies indicate that Asp 221 of Lactobacilluscasei thymidylate synthase forms a hydrogen bond network with the 2-amino and 3-imino groups of the folate [Matthews, D. A. (1990) J. Mol. Biol. 214, 937-948; Finer-Moore, J. S. (1990)Biochemistry 29, 6977-6986] that has been proposed to participate in catalysis. We prepared a complete replacement set of 19 mutants at position 221 of L. casei thymidylate synthase. Of these, the only one with sufficient activity to complement growth of a thymidylate synthase-deficient host was the Cys mutant. To further elucidate the function of the Asp 221 side chain, seven thymidylate synthase 221 mutants were studied in detail with regard to catalysis of dTMP formation and of thymidylate synthase partial reactions. Most of the mutants bound the nucleotide substrate dUMP with only moderate loss of binding affinity, indicating that the Asp side chain does not contribute to dUMP binding. Most of the mutants catalyzed the cofactor-independent dehalogenation of 5-bromodUMP; hence, the Asp side chain of TS is not essential for addition of the catalytic Cys residue to the nucleotide substrate. Mutants showed decreased affinity for the folate cofactor, but those with side chains capable of hydrogen bond formation were less severely affected. Some of the mutants were capable of forming covalent thymidylate synthase-5-fluorodUMP-methylenetetrahydrofolate complex; hence, the Asp side chain is not essential for steps leading to the covalent complex. We conclude that the hydrogen bond network between Asp 221 and the folate cofactor contributes to cofactor binding and a catalytic step after formation of the covalent ternary complex intermediate.
Chiericatti
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G
Department of Biochemistry, University of California, San Francisco 94143-0448, USA.
Santi
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DV
D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D005816:Genetic Complementation Test; D006219:Halogens / Q000378:metabolism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics; D016254:Mutagenesis, Insertional; D013763:Tetrahydrofolates / Q000378:metabolism*; D013938:Thymidine Monophosphate / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*
D003067:Coenzymes; D003856:Deoxyuracil Nucleotides; D006219:Halogens; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D001224:Aspartic Acid; D013938:Thymidine Monophosphate; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase