Interaction of deoxyuridylate with thymidylate synthetase.

Leary, Richard; Beaudette, Norman V.; Kisliuk, Roy L.

doi:10.1016/s0021-9258(19)41248-9

Cited by 81 publications

(35 citation statements)

References 17 publications

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“…The complete suppression of M1-R78 by dUMP indicates that both subunits of thymidylate synthase have similar conformations in the region including R72 and R78, which agrees with the reported symmetry of the dUMP-bound enzyme observed by Stroud and co-workers (Finer-Moore et al, 1993). However, they reported that dUMP was bound to both active sites, in contrast to previous studies by others indicating that only one dUMP was bound to each enzyme dimer (Galivan et al, 1976;Leary et al, 1975). Although an explanation for that discrepancy is not apparent, our results involving M1-R78 and M1-R72 would be consistent with dUMP binding to both subunits, which would be expected to produce identical changes in the conformation of each subunit.…”

Section: Discussionsupporting

confidence: 89%

“…Observations consistent with such changes in conformation include those from stopped-flow kinetic studies of the quenching of tryptophan fluorescence by dUMP, dTMP, and FdUMP binding, which were consistent with a two-step mechanism involving the rapid preequilibrium formation of a binary complex followed by a slower isomerization step . The reported changes in the circular dichroic spectra (Leary et al, 1975) and the protection from heat denaturation (Galivan et al, 1976) and enzymatic digestion (Galivan et al, 1977) that accompany substrate or other ligand binding are also consistent with ligand-induced conformational changes. Evidence for the induction of conformational changes in the C-terminal region of L. casei thymidylate synthase by dUMP has recently been obtained (Carreras et al, 1994).…”

supporting

confidence: 57%

“…Although there is some disagreement about the precise number of iodoacetamide molecules that react with thymidylate synthase, the number seems to be in the range of 1 (Leary et al, 1975) to 1.5 or 1.6 (Plese & Dunlap, 1977) even after incubation for periods much longer than needed for complete inactivation. It is not clear why iodoacetamide does not react with each of the catalytically essential sulfhydryl groups in the two identical subunits, unless reaction at one catalytic site induces conformational changes that block or limit reaction with the sulfhydryl group on the other subunit.…”

Section: Resultsmentioning

confidence: 99%

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Ligand-Induced Conformational Changes of Thymidylate Synthase Detected by Limited Proteolysis

Mohsen¹,

Aull²,

Payne³

et al. 1995

Biochemistry

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Limited tryptic proteolysis was used to investigate conformational changes of thymidylate synthase from Lactobacillus casei induced by ligand binding. Most of the identified sites of proteolysis were between R72 and R178, a region that includes a large loop containing residues 90-139 that is absent in thymidylate synthase from most other sources. Hydrolysis at both ends of this region was affected by the presence of dUMP. With dUMP, the preference of initial hydrolysis at the N-terminus of this region was switched from R78 to R72, and hydrolysis at R178 was retarded; the latter effect may be primarily a consequence of steric hinderance since R178 is involved in binding the phosphate moiety of dUMP. Orthophosphate had an effect similar to that of dUMP, not only in retarding hydrolysis at the phosphate binding site (R178) but also in retarding hydrolysis at R78 in favor of R72. Alkylation of the catalytically essential sulfhydryl group of thymidylate synthase by iodoacetamide also resulted in R72 being favored over R78 as a site of initial proteolysis. Its effect on hydrolysis at R178 was, as expected, less than that of dUMP or phosphate. These results indicate that dUMP binding induces conformational changes in thymidylate synthase. Phosphate binding and sulfhydryl alkylation also induce conformational changes similar to those resulting from dUMP binding. While the similarity of the proteolytic behavior of thymidylate synthase in the presence of dUMP or phosphate agrees with the report by Finer-Moore et al.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionsupporting

confidence: 89%

supporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

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Ligand-Induced Conformational Changes of Thymidylate Synthase Detected by Limited Proteolysis

Mohsen¹,

Aull²,

Payne³

et al. 1995

Biochemistry

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“…Surprisingly, a 7.9-fold excess of dCMP slowed the rate of inactivation to 9030 M-1 s-1. Previous studies by Danenberg et al (1974) and Leary et al (1975) have…”

Section: Resultsmentioning

confidence: 90%

Effects of polyoxyanions on sulfhydryl group modification of thymidylate synthetase

Lewis¹,

Munroe²,

Dunlap³

1978

Biochemistry

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“…The crystal structures of both Lactobacillus casei (Hardy et al, 1986) and E. coli TS (Matthews et al, 1990 a,b;Montfort et al, 1990) reveal that the enzyme's two identical subunits are held together by a backbone of about 30 amino acid residues with the active site in each subunit widely separated from one another despite the marked change in conformation that occurs on substrate or analogue binding. This being the case, each site should be equally active although a considerable body of evidence exists suggesting either that the two active sites contribute unequally to the enzyme's catalytic efficiency {kcx) or that only one of the two sites is active (Aull et al, 1974;Leary et al, 1975;Galivan et al, 1976a,b;Danenberg & Danenberg, 1979;Beaudette et al, 1980). Paradoxically, other studies suggest that both sites contribute equally to enzyme activity (Plese & Dunlap, 1977), particularly those involving subunit complementation wherein specific inactive mutants of L. casei TS were denatured together with urea and then renaturated by dilution, resulting in the restoration of enzyme activity (Perry et al, 1992;Pookanjanatavip et al, 1992;Carreras et al, 1994).…”

mentioning

confidence: 99%

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

Maley¹,

Pedersen-Lane²,

Changchien³

1995

Biochemistry

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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)

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Interaction of deoxyuridylate with thymidylate synthetase.

Cited by 81 publications

References 17 publications

Ligand-Induced Conformational Changes of Thymidylate Synthase Detected by Limited Proteolysis

Ligand-Induced Conformational Changes of Thymidylate Synthase Detected by Limited Proteolysis

Effects of polyoxyanions on sulfhydryl group modification of thymidylate synthetase

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

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