Complete restoration of activity to inactive mutants of Escherichia coli thymidylate synthase: evidence that E. coli thymidylate synthase is a half-the-sites activity enzyme.
Escherichia coli thymidylate synthase (TS) is a dimeric protein containing identical subunits. When R126E, an inactive mutant of this enzyme, was incubated at room temperature with other inactive mutants of E. coli, TS enzyme activity gradually reappeared. The rate of activity restoration was dependent on the mutant employed. In the case of C146W, an active site mutant, the half-time required for maximal activity restoration was about one hour, which was about 500-fold faster than that obtained with C146S. The final specific activity of the mutant mixtures, based on the concentration of R126E, was equivalent to that of the wild-type TS (WT-TS). However, when the activities of E. coli WT-TS and mutant TS mixtures were compared for their extents of renaturation following denaturation as described for Lactobacillus casei TS [Pookanjanatavip, M., Yuthavong, Y., Greene, P. J., & Santi, D. V. (1992) Biochemistry 31, 10303-10309], only about one-half of the activity of WT-TS was restored, implying that the denaturation-renaturation procedure was less efficient than allowing the native TS mutant dimers to exchange subunits. If, as proposed, subunit exchange is responsible for the observed restoration of activity to the E. coli mutant TS mixtures, it would suggest that only one active site cysteine, that provided by R126E in the dimer (R126E)-(C146W), is sufficient to yield the same kcat as WT-TS, which contains one active site cysteine in each subunit. Other mutant dimers that contain both active site cysteines such as (R126E)-(Y94A) and (R126E)-(I264Am) are also fully active, even though one of the subunits is functionally inactive.(ABSTRACT TRUNCATED AT 250 WORDS)
Maley
F
F
Wadsworth Center for Laboratories, New York State Department of Health, Albany 12201.
Pedersen-Lane
J
J
Changchien
L
L
D001665:Binding Sites; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015394:Molecular Structure; D009154:Mutation; D013940:Thymidylate Synthase / Q000737:chemistry*
D013940:Thymidylate Synthase