title: Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4.
abstract: A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995). Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS. Nine mutant ecTS enzymes that differed in sequence at position 4 were generated. Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts. Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity. Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein. The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants. Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands. However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex. Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products.
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, 730 South Main Street, Columbia, South Carolina, 29208, USA.
authors.forename: E
authors.initials: E
authors.lastname: Mahdavian
authors.forename: H T
authors.initials: HT
authors.lastname: Spencer
authors.forename: R B
authors.initials: RB
authors.lastname: Dunlap
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005468:Fluorodeoxyuridylate / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011355:Prodrugs / Q000494:pharmacology; D011487:Protein Conformation; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011355:Prodrugs; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase