Synthesis of nucleotides with specific radiolabels in ribose. Primary 14C and secondary 3H kinetic isotope effects on acid-catalyzed glycosidic bond hydrolysis of AMP, dAMP, and inosine.

Parkin, David W.; Leung, H B; Schramm, Vern L.

doi:10.1016/s0021-9258(17)42716-5

Cited by 90 publications

(107 citation statements)

References 15 publications

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“…Reactions were incubated under ambient conditions (20 °C) overnight and were terminated by the addition of sulfuric acid to a final concentration of 0.01 M. Reactions with ribose as the starting material were similar to that above except that 2 mM d -ribose was used in the place of d -glucose, and 1 unit of ribokinase was used in the place of hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphoriboisomerase (see Figure S1). All enzymes used in this synthesis were purchased from Sigma-Aldrich with the exception of ribokinase, 5-phosphoribosyl 1-pyrophosphate synthetase and adenine phosphoribosyl transferase, which were expressed and purified as previously described …”

Section: Materials and Methodsmentioning

confidence: 99%

The Transition-State Structure for Human MAT2A from Isotope Effects

Firestone

Schramm

2017

J. Am. Chem. Soc.

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Human methionine S-adenosyltransferase (MAT2A) catalyzes the formation of S-adenosylmethionine (SAM) from ATP and methionine. Synthetic lethal genetic analysis has identified MAT2A as an anticancer target in tumor cells lacking expression of 5′-methylthioadenosine phosphorylase (MTAP). Approximately 15% of human cancers are MTAP−/−. The remainder can be rendered MTAP− through MTAP inhibitors. We used kinetic isotope effect (KIE), commitment factor (Cf), and binding isotope effect (BIE) measurements combined with quantum mechanical (QM) calculations to solve the transition state structure of human MAT2A. The reaction is characterized by an advanced SN2 transition state. The bond forming from the nucleophilic methionine sulfur to the 5′-C of ATP is 2.03 Å at the transition state (bond order of 0.67). Departure of the leaving group triphosphate of ATP is well advanced and forms a 2.32 Å bond between the 5′-C of ATP and the oxygen of the triphosphate (bond order of 0.23). Interaction of MAT2A with its MAT2B regulatory subunit causes no change in the intrinsic KIEs, indicating the same transition state structure. The transition state for MAT2A is more advanced along the reaction coordinate (more product-like) than that from the near-symmetrical transition state of methionine adenosyltransferase from E. coli.

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Section: Materials and Methodsmentioning

confidence: 99%

The Transition-State Structure for Human MAT2A from Isotope Effects

Firestone

Schramm

2017

J. Am. Chem. Soc.

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“…Transition-state analysis of non-enzymatic N-glycoside hydrolysis has been reported for only one deoxynucleoside, that being 2′-deoxyadenosine monophosphate (dAMP). 26,28 The acid-catalyzed reaction, collected in 0.1 M HCl, proceeds through a stepwise mechanism in which the glycosidic bond breaks and reforms repeatedly, giving an equilibrium between the reactant and the intermediate, an adenine•oxacarbenium contact ion pair complex (CIPC). 26 Expulsion of the adenine leaving-group is catalyzed by protonation at two endocyclic nitrogens, N1 and N7, such that it departs as a monocation.…”

Section: Non-enzymatic N-glycoside Hydrolysis Of Dampmentioning

confidence: 99%

Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA

Drohat

Maiti

2014

Org. Biomol. Chem.

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DNA glycosylases remove damaged or enzymatically modified nucleobases from DNA, thereby initiating the base excision repair (BER) pathway, which is found in all forms of life. These ubiquitous enzymes promote genomic integrity by initiating repair of mutagenic and/or cytotoxic lesions that arise continuously due to alkylation, deamination, or oxidation of the normal bases in DNA. Glycosylases also perform essential roles in epigenetic regulation of gene expression, by targeting enzymatically-modified forms of the canonical DNA bases. Monofunctional DNA glycosylases hydrolyze the N-glycosidic bond to liberate the target base, while bifunctional glycosylases mediate glycosyl transfer using an amine group of the enzyme, generating a Schiff base intermediate that facilitates their second activity, cleavage of the DNA backbone. Here we review recent advances in understanding the chemical mechanism of monofunctional DNA glycosylases, with an emphasis on how the reactions are influenced by properties of the nucleobase leaving-group, the moiety that varies across the vast range of substrates targeted by these enzymes.

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“…Adenylate kinase and pyruvate kinase were purchased from Sigma-Aldrich, while ribokinase, PRPPase, and APRTase were expressed in E. coli and used as previously described. 25 The reaction was incubated at room temperature overnight, and was quenched by the addition of 110 mL of 0.1 M sulfuric acid to give a final concentration 0.01 M. The resulting 2-amino-ATP was purified from other reaction contents by reverse phase HPLC using a Kinetex C18 250 mm × 4.6 mm column eluted with 50 mM potassium phosphate and 8 mM tetrabutylammonium bisulfate (pH = 6.0), as previously described. 35 2AMTA was prepared in a 1 mL solution containing 1 mM 2-amino-ATP, 8 mM L-methionine, 50 mM tris-HCl, 50 mM potassium chloride, and 10 mM magnesium chloride.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The solution was buffered to pH = 8.0 before adding 1 unit each of ribokinase, adenine phosphoribosyltransferase (APRTase), 5′-phosphoribosylpyrophosphate synthetase (PRPPase), adenylate kinase, and pyruvate kinase to initiate the reaction. Adenylate kinase and pyruvate kinase were purchased from Sigma-Aldrich, while ribokinase, PRPPase, and APRTase were expressed in E. coli and used as previously described . The reaction was incubated at room temperature overnight, and was quenched by the addition of 110 mL of 0.1 M sulfuric acid to give a final concentration 0.01 M. The resulting 2-amino-ATP was purified from other reaction contents by reverse phase HPLC using a Kinetex C18 250 mm × 4.6 mm column eluted with 50 mM potassium phosphate and 8 mM tetrabutylammonium bisulfate (pH = 6.0), as previously described .…”

Section: Materials and Methodsmentioning

confidence: 99%

“…Chemoenzymatic and chemical synthetic schemes were developed for the production of 2AMTA. The chemoenzymatic method is based on a previous reported synthesis of AMP 25 except for substitution of adenine by 2,6-diaminopurine in the APRTase catalyzed step (Figure 2A). This synthetic approach relies on the enzymatic promiscuity of APRTase, adenylate kinase and pyruvate kinase to accept 2,6-diaminopurine and 2,6-diaminopurine nucleotides as substrates.…”

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Continuous Fluorescence Assays for Reactions Involving Adenine

et al. 2016

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5′-Methylthioadenosine phosphorylase (MTAP) and 5′-methylthioadenosine nucleosidase (MTAN) catalyze the phosphorolysis and hydrolysis of 5′-methylthioadenosine (MTA), respectively. Both enzymes have low KM values for their substrates. Kinetic assays for these enzymes are challenging, as the ultraviolet absorbance spectra for reactant MTA and product adenine are similar. We report a new assay using 2-amino-5′-methylthioadenosine (2AMTA) as an alternative substrate for MTAP and MTAN enzymes. Hydrolysis or phosphorolysis of 2AMTA forms 2,6-diaminopurine, a fluorescent and easily quantitated product. We kinetically characterize 2AMTA with human MTAP, bacterial MTANs and use 2,6-diaminopurine as a fluorescent substrate for yeast adenine phosphoribosyltransferase. 2AMTA was used as the substrate to kinetically characterize the dissociation constants for three-transition-state analogue inhibitors of MTAP and MTAN. Kinetic values obtained from continuous fluorescent assays with MTA were in good agreement with previously measured literature values, but gave smaller experimental errors. Chemical synthesis from ribose and 2,6-dichloropurine provided crystalline 2AMTA as the oxalate salt. Chemo-enzymatic synthesis from ribose and 2,6-diaminopurine produced 2-amino-S-adenosylmethionine for hydrolytic conversion to 2AMTA. Interaction of 2AMTA with human MTAP was also characterized by pre-steady-state kinetics and by analysis of the crystal structure in a complex with sulfate as a catalytically inert analogue of phosphate. This assay is suitable for inhibitor screening by detection of fluorescent product, for quantitative analysis of hits by rapid and accurate measurement of inhibition constants in continuous assays, and pre-steady-state kinetic analysis of the target enzymes.

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Synthesis of nucleotides with specific radiolabels in ribose. Primary 14C and secondary 3H kinetic isotope effects on acid-catalyzed glycosidic bond hydrolysis of AMP, dAMP, and inosine.

Cited by 90 publications

References 15 publications

The Transition-State Structure for Human MAT2A from Isotope Effects

The Transition-State Structure for Human MAT2A from Isotope Effects

Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA

Continuous Fluorescence Assays for Reactions Involving Adenine

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