Kinetic studies on drug-resistant variants of Escherichia coli thymidylatesynthase: Functional effects of amino acid substitutions at residue 4

A naturally occurring mutant of human thymidylate synthase (hTS) that conta ins a Tyr to His mutation at residue 33 was found to confer 4-fold resistan ce to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridi ne 5'-monophosphate (FdUMP), The crystal structure of hTS implicated this T yr residue in a drug resistance mechanistic role that may include both subs trate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287 , 1995). Because of the existence of a defined kinetic scheme and the devel opment of a bacterial expression vector for the overproduction of Escherich ia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS, Nine mutant ecTS enzymes that differe d 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 o f the His 4 mutant precipitated, sufficient amounts remained in the cell-fr ee extract to permit isolation to near homogeneity, Wild-type ecTS and muta nts with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nea rly 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-methylenetetrahydrofol ate (CH(2)H(4)folate) were similar for all the mutants. Dissociation consta nts for binary complex formation determined by stopped-flow spectroscopy we re similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxyt hymidylate, 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 wi th the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP i n 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 enz yme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products. (C) 1999 Academic Press.