Thermodynamic constants of the ionisation of the acid imino-group of uridine 5′-monophosphate and poly-uridylic acid

Aylward, Nigel

doi:10.1039/j29670000401

Cited by 13 publications

(14 citation statements)

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“…The computed deprotonation free energy at the N3 nitrogen of dUMP in ThyX relative to the solvent is -8.6 kcal•mol -1 (standard deviation (SD): 0.7 kcal•mol -1 ) favoring the deprotonated state of uracil. Combining this value with the experimental pK a of N3 of dUMP in a solution of 9.3 units, 31 the total deprotonation free energy is 6.8 kcal•mol -1 , which corresponds to pK a of dUMP in ThyX of 3 units in agreement with the crystallographic experiments and previous NMR measurements. 19 The unusually low pK a of uracil of dUMP in ThyX is explained by strong electrostatic interactions between the guanidinium cations of Arg199 and Arg107 and the ionized uracil ring, as shown in Figure 1.…”

Section: Thyx Recognizes the Ionized Form Of The Natural Dump Substratesupporting

confidence: 84%

“…The experimental and simulation results obtained in this work also show that the 2-OH-NQ inhibitor mimicking the natural dUMP is deprotonated at O2 in the ThyX complex and has a very similar mode of interaction with Arg199. The C8-C1 pK a value in solution of 5.5 pH units is lower than the pK a of N3 of dUMP (9.3 pH units 31 ). Thus, in the case of 2-OH naphthoquinones, there is no free energy penalty associated with the deprotonation of the 2-OH group upon binding in contrast to the natural dUMP substrate.…”

Section: Discussionmentioning

confidence: 67%

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Mechanism of Naphthoquinone Selectivity of Thymidylate Synthase ThyX

Myllykallio

Becker

Aleksandrov

2020

Biophysical Journal

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Section: Thyx Recognizes the Ionized Form Of The Natural Dump Substratesupporting

confidence: 84%

Section: Discussionmentioning

confidence: 67%

Mechanism of Naphthoquinone Selectivity of Thymidylate Synthase ThyX

Myllykallio

Becker

Aleksandrov

2020

Biophysical Journal

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“…The values of the 13 C-chemical shifts of dUMP bound to WT FDTS are nearly identical to those of unbound dUMP at pH 12, well above the pK a for N3 of dUMP (9.3 9 ), showing that N3 of dUMP is ionized when bound at pH 8 (Figure 4). The 13 C-NMR spectra at pH 8 of free dUMP and dUMP bound to WT FDTS and the variants show that R174 is largely responsible for producing the changes in the chemical shifts of the uracil of dUMP bound to FDTS.…”

Section: Resultsmentioning

confidence: 78%

“…At pH 12, dUMP in water is deprotonated (pK a for N3 is 9.3 9 ). This result suggests that N3 of dUMP is also fully ionized when bound to FDTS at pH8.…”

Section: Discussionmentioning

confidence: 99%

Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

et al. 2016

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Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2′-deoxythymidine-5′-monophosphate (dTMP) from 2′-deoxyuridine-5′-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans, and a very different mechanism. Here we report NMR evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogs also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.

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“…The pKa of the proton at N3 of dUMP was experimentally determined to be ∼9.0−9.5. 55 The hydride acceptor is likely to have this proton still present. This prediction is confirmed with the QM/MM free energy surface shown in Figure 5 that explores the equilibrium between N3(H) and O4(H) tautomers.…”

Section: Resultsmentioning

confidence: 99%

Experimental and Computational Studies Delineate the Role of Asparagine 177 in Hydride Transfer for E. coli Thymidylate Synthase

et al. 2018

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Thymidylate synthase (TSase), an enzyme responsible for the de novo biosynthesis of 2’-deoxythymidine 5’-monophosphate (thymidylate, dTMP) necessary for DNA synthesis, has been a drug target for decades. TSase is a highly conserved enzyme across species ranging from very primitive organisms to mammals. Among the many conserved active site residues, an asparagine (N177, using Escherichia coli residues numbering) appears to make direct hydrogen bonds with both the C4=O4 carbonyl of the 2’-deoxyuridine 5’-monophosphate (uridylate, dUMP) substrate and its pyrimidine ring’s N3. Recent studies have reassessed the TSase catalytic mechanism, focusing on the degree of negative charge accumulation at the O4 carbonyl of the substrate during two critical H-transfers - a proton abstraction and a hydride transfer. To obtain insights into the role of this conserved N177 on the hydride transfer, we examined its aspartic acid (D) and serine (S) mutants - each of which is expected to alter hydrogen bonding and charge stabilization around the C4=O4 carbonyl of the 2’-deoxyuridine 5’-monophosphate (uridylate, dUMP) substrate. Steady-state kinetics, substrate binding order studies and temperature-dependency analysis of intrinsic KIEs for the hydride transfer step of the TSase catalytic cycle suggest the active site of N177D is not precisely organized for that step. A smaller disruption was observed for N177S, which could be rationalized by partial compensation by water molecules and rearrangement of other residues toward preparation of the system for the hydride transfer under study. These experimental findings are qualitatively mirrored by QM/MM computational simulations, thereby shedding light on the sequence and synchronicity of steps in the TSase-catalyzed reaction. This information could potentially inform the design of mechanism-based drugs targeting this enzyme.

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Thermodynamic constants of the ionisation of the acid imino-group of uridine 5′-monophosphate and poly-uridylic acid

Cited by 13 publications

References 0 publications

Mechanism of Naphthoquinone Selectivity of Thymidylate Synthase ThyX

Mechanism of Naphthoquinone Selectivity of Thymidylate Synthase ThyX

Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

Experimental and Computational Studies Delineate the Role of Asparagine 177 in Hydride Transfer for E. coli Thymidylate Synthase

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