Limits...
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
HRMS of5-hydroxymethyl-dUMP isolated from the acid-quenched FDTSreactions in H2O and D2O.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4121000&req=5

fig2: HRMS of5-hydroxymethyl-dUMP isolated from the acid-quenched FDTSreactions in H2O and D2O.

Mentions: Recently, we isolated and characterized a derivative of anintermediate(s)in FDTS-catalyzed thymidylate synthesis, in rapid acid-quenching experimentsconducted at room temperature.11 This trappedspecies (5-hydroxymethyl-dUMP, or 5-HM-dUMP, Scheme 2) already contains the methylene of CH2H4fol. 5-HM-dUMP is consistent with either of the two proposed mechanismsfor FDTS (Scheme 1) and does not distinguishbetween them. In the current work, in an attempt to differentiatebetween the mechanisms, we repeated acid-quenching experiments withFDTS reactions taking place in deuterated water (D2O).The reaction conditions were kept the same as in the quenching studiesin H2O,11 except all reactantsand buffers were exchanged into D2O by cycles of lyophilizationand resuspension in heavy water (99.9% D). In D2O, allexchangeable hydrogens, including the N5 hydrogen of the reduced flavinto be transferred to the uracil moiety, are exchanged with their heavierisotopes. Thus, we anticipated that, if the hydride from the flavinis transferred to dUMP before the methylene (Scheme 1a), then a portion of acid-trapped 5-HM-dUMP would be deuterated,i.e., one mass unit heavier than in the reactions conducted in H2O (Scheme 2a). On the other hand, onthe basis of the mechanism proposed in Scheme 1b, no effect on the mass of the trapped intermediate was expected(Scheme 2b). As shown in Figure 2, no deuterium enrichment is observed in 5-HM-dUMP isolatedin the D2O experiment. Importantly, all dTMP product presentin D2O reactions was singly deuterated, eliminating thepossibility of protium contamination in the experiment and in accordancewith previously reported deuterium incorporation into dTMP.10


Substrate activation in flavin-dependent thymidylate synthase.

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

HRMS of5-hydroxymethyl-dUMP isolated from the acid-quenched FDTSreactions in H2O and D2O.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: HRMS of5-hydroxymethyl-dUMP isolated from the acid-quenched FDTSreactions in H2O and D2O.
Mentions: Recently, we isolated and characterized a derivative of anintermediate(s)in FDTS-catalyzed thymidylate synthesis, in rapid acid-quenching experimentsconducted at room temperature.11 This trappedspecies (5-hydroxymethyl-dUMP, or 5-HM-dUMP, Scheme 2) already contains the methylene of CH2H4fol. 5-HM-dUMP is consistent with either of the two proposed mechanismsfor FDTS (Scheme 1) and does not distinguishbetween them. In the current work, in an attempt to differentiatebetween the mechanisms, we repeated acid-quenching experiments withFDTS reactions taking place in deuterated water (D2O).The reaction conditions were kept the same as in the quenching studiesin H2O,11 except all reactantsand buffers were exchanged into D2O by cycles of lyophilizationand resuspension in heavy water (99.9% D). In D2O, allexchangeable hydrogens, including the N5 hydrogen of the reduced flavinto be transferred to the uracil moiety, are exchanged with their heavierisotopes. Thus, we anticipated that, if the hydride from the flavinis transferred to dUMP before the methylene (Scheme 1a), then a portion of acid-trapped 5-HM-dUMP would be deuterated,i.e., one mass unit heavier than in the reactions conducted in H2O (Scheme 2a). On the other hand, onthe basis of the mechanism proposed in Scheme 1b, no effect on the mass of the trapped intermediate was expected(Scheme 2b). As shown in Figure 2, no deuterium enrichment is observed in 5-HM-dUMP isolatedin the D2O experiment. Importantly, all dTMP product presentin D2O reactions was singly deuterated, eliminating thepossibility of protium contamination in the experiment and in accordancewith previously reported deuterium incorporation into dTMP.10

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