Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation.
The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.
Carreras
C W
CW
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.
Climie
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SC
Santi
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DV
D002384:Catalysis; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007065:Idoxuridine / Q000031:analogs & derivatives / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000201:enzymology; D016296:Mutagenesis; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013763:Tetrahydrofolates / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000096:biosynthesis*; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D013438:Sulfhydryl Compounds; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C056075:iododeoxyuridylate; D013938:Thymidine Monophosphate; C017089:bromodeoxyuridylate; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase; D007065:Idoxuridine