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Substrate activation in flavin-dependent thymidylate synthase.

Mishanina TV, Corcoran JM, Kohen A - J. Am. Chem. Soc. (2014)

Bottom Line: The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors.Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques.Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Iowa , Iowa City, Iowa 52242-1727, United States.

ABSTRACT
Thymidylate is a critical DNA nucleotide that has to be synthesized in cells de novo by all organisms. Flavin-dependent thymidylate synthase (FDTS) catalyzes the final step in this de novo production of thymidylate in many human pathogens, but it is absent from humans. The FDTS reaction proceeds via a chemical route that is different from its human enzyme analogue, making FDTS a potential antimicrobial target. The chemical mechanism of FDTS is still not understood, and the two most recently proposed mechanisms involve reaction intermediates that are unusual in pyrimidine biosynthesis and biology in general. These mechanisms differ in the relative timing of the reaction of the flavin with the substrate. The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors. Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques. Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data. These findings and the newly proposed mechanism add an important piece to the puzzle of the mechanism of FDTS and suggest a new class of intermediates that, in the future, may serve as targets for mechanism-based design of FDTS-specific inhibitors.

Show MeSH
Acid Trapping of the Proposed Intermediatesin the Reaction withDeuterium-Labeled Flavin (FADD2)Formationof 5-HM-dUMP in (a)requires oxidation of the reduced intermediates at C6, i.e., lossof a hydron (H+ or D+) and two electrons. Dueto an isotope effect on this nonenzymatic oxidation, the majorityof 5-HM-dUMP is expected to be deuterated. Molecular oxygen has beenproposed as the oxidant,11 since quenchedreactions are exposed to oxygen during quenching.
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sch2: Acid Trapping of the Proposed Intermediatesin the Reaction withDeuterium-Labeled Flavin (FADD2)Formationof 5-HM-dUMP in (a)requires oxidation of the reduced intermediates at C6, i.e., lossof a hydron (H+ or D+) and two electrons. Dueto an isotope effect on this nonenzymatic oxidation, the majorityof 5-HM-dUMP is expected to be deuterated. Molecular oxygen has beenproposed as the oxidant,11 since quenchedreactions are exposed to oxygen during quenching.


Substrate activation in flavin-dependent thymidylate synthase.

Mishanina TV, Corcoran JM, Kohen A - J. Am. Chem. Soc. (2014)

Acid Trapping of the Proposed Intermediatesin the Reaction withDeuterium-Labeled Flavin (FADD2)Formationof 5-HM-dUMP in (a)requires oxidation of the reduced intermediates at C6, i.e., lossof a hydron (H+ or D+) and two electrons. Dueto an isotope effect on this nonenzymatic oxidation, the majorityof 5-HM-dUMP is expected to be deuterated. Molecular oxygen has beenproposed as the oxidant,11 since quenchedreactions are exposed to oxygen during quenching.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4121000&req=5

sch2: Acid Trapping of the Proposed Intermediatesin the Reaction withDeuterium-Labeled Flavin (FADD2)Formationof 5-HM-dUMP in (a)requires oxidation of the reduced intermediates at C6, i.e., lossof a hydron (H+ or D+) and two electrons. Dueto an isotope effect on this nonenzymatic oxidation, the majorityof 5-HM-dUMP is expected to be deuterated. Molecular oxygen has beenproposed as the oxidant,11 since quenchedreactions are exposed to oxygen during quenching.
Bottom Line: The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors.Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques.Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Iowa , Iowa City, Iowa 52242-1727, United States.

ABSTRACT
Thymidylate is a critical DNA nucleotide that has to be synthesized in cells de novo by all organisms. Flavin-dependent thymidylate synthase (FDTS) catalyzes the final step in this de novo production of thymidylate in many human pathogens, but it is absent from humans. The FDTS reaction proceeds via a chemical route that is different from its human enzyme analogue, making FDTS a potential antimicrobial target. The chemical mechanism of FDTS is still not understood, and the two most recently proposed mechanisms involve reaction intermediates that are unusual in pyrimidine biosynthesis and biology in general. These mechanisms differ in the relative timing of the reaction of the flavin with the substrate. The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors. Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques. Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data. These findings and the newly proposed mechanism add an important piece to the puzzle of the mechanism of FDTS and suggest a new class of intermediates that, in the future, may serve as targets for mechanism-based design of FDTS-specific inhibitors.

Show MeSH