- Enzyme architecture: Deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5′-monophosphate decarboxylase
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The mechanism for activation of orotidine 5′-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters kcat and K m for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-d-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (kcat)obs for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (Km)obs.
- Goldman, Lawrence M.,Amyes, Tina L.,Goryanova, Bogdana,Gerlt, John A.,Richard, John P.
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- Phosphorylation, oligomerization and self-assembly in water under potential prebiotic conditions
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Prebiotic phosphorylation of (pre)biological substrates under aqueous conditions is a critical step in the origins of life. Previous investigations have had limited success and/or require unique environments that are incompatible with subsequent generation of the corresponding oligomers or higher-order structures. Here, we demonstrate that diamidophosphate (DAP) - a plausible prebiotic agent produced from trimetaphosphate - efficiently (amido)phosphorylates a wide variety of (pre)biological building blocks (nucleosides/tides, amino acids and lipid precursors) under aqueous (solution/paste) conditions, without the need for a condensing agent. Significantly, higher-order structures (oligonucleotides, peptides and liposomes) are formed under the same phosphorylation reaction conditions. This plausible prebiotic phosphorylation process under similar reaction conditions could enable the systems chemistry of the three classes of (pre)biologically relevant molecules and their oligomers, in a single-pot aqueous environment.
- Gibard, Clémentine,Bhowmik, Subhendu,Karki, Megha,Kim, Eun-Kyong,Krishnamurthy, Ramanarayanan
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- 5’-Phosphorylation Increases the Efficacy of Nucleoside Inhibitors of the DNA Repair Enzyme SNM1A
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Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5’–3’ exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5’-position, as well as zinc-binding groups at the 3’-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5’-phosphate dramatically increased the potency of the inhibitors.
- Berney, Mark,Fay, Ellen M.,Manoj, Manav T,McGouran, Joanna F.
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supporting information
(2022/01/13)
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- Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics
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Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (kcat = 21 s-1 and KM = 5 μM) and the molar enthalpy (ΔHapp = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 μm and 29 μM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.
- Poduch, Ewa,Bello, Angelica M.,Tang, Sishi,Fujihashi, Masahiro,Pai, Emil F.,Kotra, Lakshmi P.
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p. 4937 - 4945
(2007/10/03)
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- A solid phase reagent for the capture phosphorylation of carbohydrates and nucleosides.
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[figure: see text] A 1% cross-linked divinylbenzene-polystyrene copolymer, containing cyanoethoxy N,N-diisopropylamine phosphine was prepared as a phosphitylating agent. The polymer-bound phosphitylated precursor was subjected to reaction with alcohols in the presence of 1H-tetrazole to produce the corresponding polymer-bound phosphite triesters. These were then oxidized with tert-butyl hydroperoxide to give the polymer-bound monophosphate triesters. Removal of cyanoethoxy on the resin with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) followed by basic cleavage of the p-hydroxybenzyl linker products yielded monophosphate derivatives.
- Parang,Fournier,Hindsgaul
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p. 307 - 309
(2007/10/03)
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- Practical Synthesis of 5-Phospho-D-ribosyl α-1-pyrophosphate (PRPP): Enzymatic Routes from Ribose 5-Phosphate or Ribose
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This paper describes enzymatic syntheses of 5-phospho-D-ribosyl α-1-pyrophosphate (PRPP) on a 75-mmol scale.The reactions used PAN-immobilized PRPP synthetase as catalyst with in situ ATP-cofactor regeneration.In one procedure pure r-5-P was used as a starting material; in a second, r-5-P was synthesized by ribokinase-catalyzed phosphorilation of D-ribose and used in situ.The potential for use of PRPP as a starting material for the preparation of nucleotides was demonstrated by an enzymatic synthesis of UMP.This paper also describes several methods for the preparation of r-5-P: acid-catalyzed hydrolysis of AMP, acid-catalyzed hydrolysis of crude mononucleotide mixture obtained by digestion of RNA, chemical synthesis from D-ribose, and ribokinase-catalyzed synthesis from D-ribose.Procedures are described for the isolation of PRPP synthetase (from Salmonella typhimurium) and ribokinase (from Lactobacillus plantarum) and for immobilization of these enzymes in PAN.
- Gross, Akiva,Abril, Obsidiana,Lewis Jerome M.,Geresh, Shimona,Whitesides, George M.
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p. 7428 - 7435
(2007/10/02)
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- SYNTHETIC NUCLEOSIDES AND NUCLEOTIDES. XV. 5-DIMETHYLAMINO-2-OXIDOISOQUINOLIN-1-YL DIAZOMETHANE: A NOVEL WATER-SOLUBLE FLUORESCENT LABELLING AGENT FOR NUCLEOTIDES
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A novel fluorescent labelling agent, 5-dimethylamino-2-oxidoisoquinolin-1-yl diazomethane (3) was designed and synthesized for the fluorescent labelling of the phosphate moiety of nucleotides and nucleic acids.Starting from 1-cyano-5-nitroisoquinoline (4), 1-carboxy-5-nitroisoquinoline (5) was obtained after hydrolysis with hydrochloric acid.Esterification of 5 with methanol in the presence of sulfuric acid afforded 1-methoxycarbonyl-5-nitroisoquinoline (6).Catalytic hydrogenation of 6 followed by treatment with formic acid-acetic anhydride gave the 5-formamido derivative (8).Methylation of 8 with methyl iodide in the presence of sodiu m hydride afforded the 5-N-methylformamido derivative (9).Reduction of both the ester group and formyl group with aluminum hydride followed by treatment with chloranil and acetic anhydride provided 1-acetoxymethyl-5-dimethylaminoisoquinoline (11).N-Oxidation of 11 with m-chloroperbenzoic acid followed by selective removal of the oxido group at the 5-position by reaction with carbon disulfide afforded 1-acetoxymethyl-5-dimethylaminoisoquinoline-2-oxide (13).After deacylation of 13, selenium dioxide oxidation of the hydroxymethyl group followed by reaction with p-toluenesulfonyl hydrazide gave 5-dimethylamino-1-formylisoquinoline-2-oxide p-toluenesulfonyl hydrazone (16).On treatment of 16 with sodium ethoxide, the desired compound (3) was obtained.Reaction of 3 with p-nitrobenzoic acid gave the crystalline p-nitrobenzoyl ester.Treatment of uridine 5'-phosphate with 3 gave uridine 5'-(5-dimethylamino-2-oxidoisoquinolin-1-yl)methylphosphate (17).This labelled nucleotide was highly fluorescent, with an excitation maximum of 353 nm and an emission maximum at 523 nm.The fluorescence characteristics of 17 were compared with those of the model compound (13) and uridine 5'-(5-dimethylaminoisoquinolin-1-yl) methylphosphate (18).Keywords- fluorescent labelling agent; fluorescent labelling of nucleotides; 5-dimethylamino-2-oxidoisoquinolin-1-yl diazomethane; synthesis; reaction with uridine 5'-phosphate; fluorescence properties;uridine 5'-(5-dimethylaminoisoquinolin-1-yl)methylphosphate; uridine 5'-(5-dimethylamino-2-oxidoisoquinolin-1-yl)methylphosphate
- Nishimura, Shigeko,Saneyoshi, Mineo
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p. 1695 - 1703
(2007/10/02)
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