Standard Free Energy Change for the Hydrolysis of the .alpha.,.beta.-Phosphoanhydride Bridge in ATP

Frey, Perry A.; Arabshahi, Abolfazl

doi:10.1021/bi00036a001

Cited by 46 publications

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“…This value should be compared to Ϫ47 kJ mol Ϫ1 for the hydrolysis of the ␣,␤-phosphoanhydride bond of ATP (34). Consistently, the equilibrium constant for the diphosphoryl transfer reaction of PRPP synthase has been determined to be 29 (35).…”

mentioning

confidence: 87%

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Hove‐Jensen

Andersen

Kilstrup

et al. 2017

Microbiol Mol Biol Rev

169

187

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SUMMARY Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

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mentioning

confidence: 87%

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Hove‐Jensen

Andersen

Kilstrup

et al. 2017

Microbiol Mol Biol Rev

169

187

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“…Another factor favoring the adenylylation of DNA ligase is the energy of the ␣,␤-phosphoanhydride linkage in ATP. The standard free energy of hydrolysis has recently been found to be Ϫ10.9 kcal mol Ϫ1 (14), which is significantly more negative than the Ϫ7.8 kcal mol Ϫ1 for hydrolysis of the ␤,␥-phosphoanhydride linkage (21). The combined effects of the high energy ␣,␤-phosphoanhydride linkage in ATP and the low value of pK a for Lys 159 in T4 DNA ligase facilitate the formation of the intermediate AMP ligase.…”

Section: Table I Standard Free Energy Change For Reactions Of Dna Ligmentioning

confidence: 98%

“…The resulting phosphoanhydride linkage is a P 1 ,P 2 -dialkyl diphosphate, and as such can be expected to display a similar value of ⌬GЈ°to that for the hydrolysis of UDP-glucose to UMP and glucose-1-phosphate, which is Ϫ10.3 kcal mol Ϫ1 (14). This is 2.9 kcal mol Ϫ1 less negative than ⌬GЈ°for the hydrolysis of adenylyl-DNA ligase, so that the transfer of the AMP group from the adenylylated enzyme to nicked DNA appears to be spontaneous.…”

Section: Table I Standard Free Energy Change For Reactions Of Dna Ligmentioning

confidence: 98%

“…However, the adenosine-5Ј-phosphoramidate formed in the first step is expected to be a high energy intermediate. The standard free energy change for the hydrolysis of E-Lys-NH-AMP (nucleoside-5Ј-phosphoramidate) has not been known; however, it could be calculated from the equilibrium constant for adenylylation of the enzyme at pH 7.0 and the standard free energy change of Ϫ10.9 kcal mol Ϫ1 for the hydrolysis of ATP to AMP and PP i (14). In this study, we report the equilibrium constants for the adenylylation of T4 DNA ligase at ten different pH values, the apparent pK a for dissociation of the protonated lysine residue of the T4 DNA ligase, and the standard free energy change for the hydrolysis of adenylylated T4 DNA ligase.…”

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confidence: 99%

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Standard Free Energy for the Hydrolysis of Adenylylated T4 DNA Ligase and the Apparent pK of Lysine 159

Arabshahi¹,

Frey²

1999

Journal of Biological Chemistry

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Equilibrium constants for the adenylylation of T4 DNA ligase have been measured at 10 pH values. The values, when plotted against pH, fit a titration curve corresponding to a pK a of 8.4 ؎ 0.1. The simplest interpretation is that the apparent pK a is that of the 6-amino group of the AMP-accepting residue Lys 159 . Based on the pH dependence of the equilibrium constants, the value at pH 7.0 is 0.0213 at 25°C, corresponding to ⌬G o ‫؍‬ ؉2.3 kcal mol ؊1 . From this value and the standard free energy change of -10.9 kcal mol ؊1 for the hydrolysis of ATP to AMP and PP i , we calculate that ⌬G o for the hydrolysis of the adenylyl-DNA ligase is -13.2 kcal mol ؊1 . The presence of conserved basic amino acid residues in the catalytic domain, which are proximal to the active site in the homologous catalytic domain of T7 DNA ligase, suggests that the pK a of Lys 159 is perturbed downward by the electrostatic effects of nearby positively charged amino acid side chains. The lower than normal pK a 8.4 compared with 10.5 for the 6-amino group of lysine and the high energy of the ␣,␤-phosphoanhydride linkage in ATP significantly facilitate adenylylation of the enzyme.DNA ligases catalyze phosphodiester-bond formation between the adjacent 5Ј-phosphate and 3Ј-hydroxyl ends of the nicked DNA chains (1, 2). DNA ligases were first identified in 1967 (3-8) and shown to play essential roles in DNA replication, repair, and recombination. Recently, interest in DNA ligases has been expanded by new information regarding the connections between human cancers and DNA repair systems (9, 10). The two types of DNA ligases are ATP-dependent and NAD ϩ -dependent, respectively. ATP-dependent ligases are from eukaryotic cells, certain prokaryotes, and bacteriophages of the T series; NAD ϩ -dependent enzymes are from other prokaryotic cells. DNA ligases require a divalent cation for activity, and the optimum concentration of Mg 2ϩ for the T4 DNA ligase is 10 mM (2). Molecular weights of DNA ligases vary considerably, from 41 kDa in T7 to more than 100 kDa for the mammalian DNA ligases. The T4 DNA ligase is a single polypeptide with the molecular weight of 68,000 (11). The crystal structure of T4 DNA ligase has not been determined; however, that of T7 DNA ligase has been solved at 2.6 Å (10).DNA ligation proceeds in three reversible steps by a ping pong kinetic mechanism (2, 12) as shown in Eqs. 1a-1c for ATP-dependent ligases.E-Lys-NH 2 ϩ ATP 7 E-Lys-NH-AMP ϩ PP i (Eq. 1a)E-Lys-NH-AMP ϩ DNA-5Ј-OP 7 E-Lys-NH 2 ϩ DNA-5Ј-OP-AMP (Eq. 1b)DNA ligase reacts with ATP in the first step to displace PP i and generate an AMP enzyme (adenylyl enzyme) complex in which the AMP is linked to the 6-amino group of a lysine residue of the enzyme through a phosphoramide bond (13). The AMP enzyme then binds nicked DNA and transfers the AMP group to the 5Ј-phosphoryl group in the nick. The resulting adenosine-5Ј-pyrophosphoryl moiety in the nick activates the phosphate group at the 5Ј terminus of the DNA by supplying AMP as a good leaving group. The final step is a nucle...

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“…The value used for the standard free energy of hydrolysis of ATP to form AMP and PP i was recently re-evaluated by Frey and Arabshahi (8). The value reported of Ϫ10.9 kcal mol Ϫ1 is significantly higher than the values of Ϫ7.7 to Ϫ8.4 kcal mol Ϫ1 listed in many biochemistry textbooks.…”

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confidence: 99%

Determination of the Free-Energy Change for Repair of a DNA Phosphodiester Bond

Dickson¹,

Burns²,

Richardson

2000

Journal of Biological Chemistry

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The repair of phosphodiester bonds in nicked DNA is catalyzed by DNA ligases. Ligation is coupled to cleavage of a phosphoanhydride bond in a nucleotide cofactor resulting in a thermodynamically favorable process. A free energy value for phosphodiester bond formation was calculated using the reversibility of the T4 DNA ligase reaction. The relative number of DNA nicks to phosphodiester bonds in a circular plasmid DNA, formed during this reaction at fixed concentrations of ATP to AMP and PP i , was quantified. At 25°C, pH 7, the equilibrium constant (K eq ) for the ligation reaction is 3.89 ؋ 10 4 M. This value corresponds to a standard free energy (⌬G°) of ؊6.3 kcal mol ؊1 . By subtracting the known energy contribution due to hydrolysis of ATP to AMP and PP i , ⌬G° for the hydrolysis of a DNA phosphodiester bond is ؊5.3 kcal mol ؊1 .The repair of a single-strand break in DNA involves synthesis of a phosphodiester bond by condensation of a 5Ј-phosphoryl group on one nucleotide with a 3Ј-hydroxyl group on an adjacent nucleotide. DNA ligases catalyze this reaction by coupling condensation to cleavage of a phosphoanhydride bond from either NAD ϩ or ATP (1). The overall reaction favors phosphodiester bond formation. However, a quantitative value for this energy change has never been reported, nor has the standard free energy for the hydrolysis of a phosphodiester bond in DNA been determined. T4 DNA ligase, as well as ligases from eukaryotic and some prokaryotic cells, uses ATP as a co-substrate to catalyze DNA repair (2). The DNA ligase catalyzed reaction is reversible, as first demonstrated for Escherichia coli DNA ligase by conversion of a supercoiled circular DNA to a relaxed circular form (3). The reaction scheme for ATP-dependant DNA ligases is illustrated in Scheme 1.T4 DNA ligase, acting on a circular DNA template, was used in a DNA nicking assay to calculate an equilibrium constant (K eq ) for the ligation reaction. From this value, the corresponding standard free energies (⌬G°Ј) of both the ligation and phosphodiester bond hydrolysis reactions were determined. EXPERIMENTAL PROCEDURES T4 DNA ligase was purchased from New England Biolabs, Inc. and had a specific activity of 10 6 NEB units/mg. ATP was from Roche Molecular Biochemicals, and AMP was from Sigma. The concentrations of the adenine nucleotides were determined spectrophotometrically using ⑀ 259 ϭ 1.54 ϫ 10 4 . Na 4 P 2 O 7 -10 H 2 O was from Fisher Scientific. Ethidium bromide was from Sigma. All other reagents were ACS certified or better.The two circular plasmid DNAs used were as follows: the 3. et al. (6). This method involved concentrating the supercoiled DNA by centrifugation through a cesium chloride density gradient containing ethidium bromide. Residual ethidium bromide was subsequently removed from the DNA preparation by extraction with isobutanol, and the concentration of the DNA was determined as described by Dillon et al. (6). Linear DNA was formed by digestion with HindIII (New England Biolabs). Ligation reactions were performed in a sta...

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Standard Free Energy Change for the Hydrolysis of the .alpha.,.beta.-Phosphoanhydride Bridge in ATP

Cited by 46 publications

References 20 publications

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Standard Free Energy for the Hydrolysis of Adenylylated T4 DNA Ligase and the Apparent pK of Lysine 159

Determination of the Free-Energy Change for Repair of a DNA Phosphodiester Bond

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