Properties of a defined mutant of Escherichia coli thymidylate synthase.
A mutant of Escherichia coli thymidylate synthase (F3-TS), resulting from the replacement of a tyrosine for a cysteine 50 amino acids from the amino-terminal end, has been purified to homogeneity and found to contain less than 0.2% of the activity of the native enzyme (thyA-TS). Although this protein formed a ternary complex with 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and 5,10-methylenetetrahydrofolate, like the native enzyme, the extent of complex formation was significantly impaired as determined by equilibrium dialysis and circular dichroism. Thus, unlike the native enzyme, where 2 mol of FdUMP were present in each mole of ternary complex, F3-TS contained less than 1 mol of FdUMP/mol of ternary complex. Similarly, the binding of dUMP by F3-TS was greatly diminished relative to thyA-TS, but its binding as well as that of FdUMP could be improved by the presence of either the folate substrate or a tight binding folate analogue, 10-propargyl-5,8-dideazafolate (PDDF). However, despite the fact that PDDF enhanced the binding of FdUMP and dUMP to F3-TS, the binding of PDDF to the mutant enzyme was also greatly impaired. This contrasts with the native enzyme, which, under the same conditions, bound about 2 mol of PDDF/mol of enzyme in the presence or absence of either FdUMP or dUMP. Circular dichroism analyses with PDDF in the presence of dUMP or FdUMP yielded analogous results, but the effects were less dramatic than those obtained by equilibrium dialysis. Evidence in support of a structural difference between thyA-TS and F3-TS was obtained by demonstrating that the latter protein was 15-fold slower in forming a ternary complex with dUMP and PDDF than the former and that the mutant enzyme was less stable than the native enzyme.
Maley
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Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Maley
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D000596:Amino Acids / Q000032:analysis; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D009154:Mutation; D010449:Peptide Mapping; D011799:Quinazolines / Q000378:metabolism; D013329:Structure-Activity Relationship; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics*; D014443:Tyrosine
D000596:Amino Acids; D011799:Quinazolines; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D014443:Tyrosine; C031662:CB 3717; D005492:Folic Acid; D013940:Thymidylate Synthase; D003545:Cysteine