Identification of the Purine Nucleotides a and b as the 2'- and 3'-Phosphoribosides, Respectively<sup>1</sup>

Khym, Joseph X.; Cohn, Waldo E.

doi:10.1021/ja01636a025

Cited by 30 publications

(6 citation statements)

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“…By NMR, the ratio of 2′- to 3′-linked ester was determined to be approximately 3:7, in agreement with previous measurements for similar compounds(26, 36). Therefore, the 3′-oxygen-18 effect is primarily derived from being the leaving group, while the 2′-oxygen-18 effect is primarily from being vicinal to the leaving group.…”

Section: Resultssupporting

confidence: 90%

Transition States of Uncatalyzed Hydrolysis and Aminolysis Reactions of a Ribosomal P-Site Substrate Determined by Kinetic Isotope Effects

et al. 2010

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The ester bond of peptidyl-tRNA undergoes nucleophilic attack both in solution and catalyzed by the ribosome. To characterize the uncatalyzed hydrolysis reaction -a model of peptide release -the transition state structure for hydrolysis of a peptidyl-tRNA mimic was determined. Kinetic isotope effects were measured at several atoms that potentially undergo a change in bonding at the transition state. Large kinetic isotope effects of carbonyl oxygen-18 and α-deuterium substitutions on uncatalyzed hydrolysis indicate the transition state is nearly tetrahedral. Kinetic isotope effects were also measured for aminolysis by hydroxylamine to study a reaction similar to peptide bond formation. In contrast to hydrolysis, the large leaving group oxygen-18 isotope effect indicates the C-O3′ bond has undergone significant scission in the transition state. The smaller carbonyl oxygen-18 and α-deuterium effects are consistent with a later transition state. The assay developed here can also be used to measure isotope effects for the ribosome-catalyzed reactions. These uncatalyzed reactions serve as a basis to determine what aspects of the transition states are stabilized by the ribosome to achieve a rate enhancement.The ribosome catalyzes two chemical reactions during protein synthesis. During initiation and elongation, peptide bonds are formed by the nucleophilic attack of an aminoacyl-tRNA in the A-site on peptidyl-tRNA in the P-site. During termination, water acts as the nucleophile resulting in the release of the peptide chain from tRNA. The ribosome must be capable of selectively switching between these two catalytic activities, since hydrolysis during elongation would produce truncated proteins, and peptide bond formation in place of termination would result in read-through of stop codons.In the last decade, a wealth of structural information has been obtained to complement biochemical study of catalysis by the ribosome. Structures of the 50S ribosome demonstrated that the active site for peptide bond formation is composed entirely of RNA(1,2). Of the functional groups present in the active site, two have been identified as important for catalysis of peptide bond formation: the 2′-hydroxyl of A2451 in 50S RNA contributes about 10-fold (3), and the 2′-hydroxyl of the terminal adenosine of peptidyl-tRNA, which is adjacent to the leaving group, contributes at least 10 6 -fold(4). Brönsted(5) and kinetic isotope effect studies (6) indicate that the nucleophilic nitrogen is neutral in the transition state despite at least partial peptide bond formation. More recently, structures of 70S ribosomes bound to either release † This research was supported by NIH grant GM54839 (to SAS), NIH post-doctoral fellowship GM079980 (to DAH), and a Brown-Coxe fellowship (to VS).*Author to whom correspondence should be addressed: NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript factor 1 (7) or release factor 2 (8,9) have shown the location of the conserved GGQ motif in the active site for peptide release. Mut...

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Section: Resultssupporting

confidence: 90%

Transition States of Uncatalyzed Hydrolysis and Aminolysis Reactions of a Ribosomal P-Site Substrate Determined by Kinetic Isotope Effects

et al. 2010

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“…The difference in free energy between isomers of diol esters is slight, as might be expected. In adenosine derivatives, the equilibrium ratio of adenosine-3 '-phosphate to adenosine-2'-phosphate is 2.5(Khym and Cohn, 1954). Similarly, the equilibrium ratio of the 3' to the 2' amino acid esters of adenosine and of s-RNA indicated by the present findings is 2.0.…”

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confidence: 67%

On the Site of Esterification of Amino Acids to Soluble RNA^*

Wolfenden¹,

Rammler²,

Lipmann³

1964

Biochemistry

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Aminoacyladenosine was isolated from aminoacy 1-RN A by ribonuclease digestion and ion exclusion on DEAE-cellulose. To determine the position of esterification of the amino acid, 2,3-dihydropyran was used to block the unoccupied hydroxyl groups of the aminoacyl nucleoside, the amino acid was removed in mild alkali, and the hydroxyl group originally occupied by the amino acid was phosphory lated. Each step in the reaction sequence was carefully evaluated with model compounds. After removal of the protecting groups, 2' and 3' adenylic acids were isolated in a molar ratio of 1:2. A study of the comparative rates of acyl migration and hydrolysis in esters of glycerol, as models, shows that acyl equilibration between a and ß positions is rapid, exceeding the rate of hydrolysis by a factor of more than 6000 in the neutral and alkaline range. Tentative extrapolation to aminoacy 1-RN A suggests that aminoacyl 2' and 3' esters may be in base-catalyzed tautomeric equilibrium at pH values near neutrality.The binding of amino acids to soluble RNA (s-RNA) (Hoagland et al., 1957) has been shown to involve attachment to the 2' or 3' hydroxyl group of the adenosine residue which terminates the nucleic acid chain (Zachau et al., 1958;Preiss et al., 1959). Attachment by ester linkage was inferred from the ease of hydroxylaminolysis and alkaline hydrolysis of these compounds * This work was supported in part by a grant from the National Science Foundation.

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“…Thus the designation of uridylic acids a and b as the 2'and 3'-isomers, respectively, which has hitherto rested on the physicochemical properties of the cytidylic acids29 and the deamination linking of a cytidylic to a uridylic and of b to b30•31 is now confirmed by direct isolation and identification of the ribose phosphate moiety as with the purine nucleotides. [22][23] Thymidylic acid was reduced under the same conditions, but at a slower rate, as were the ribotides. Consumption of hydrogen, disappearance of spectrum and appearance of reactivity in the diphenylamine reaction were used as the criteria of reduction.…”

Section: Resultsmentioning

confidence: 99%

The Catalytic Hydrogenation of Pyrimidine Nucleosides and Nucleotides and the Isolation of their Ribose and Respective Ribose Phosphates^1,2

Cohn¹,

Doherty²

1956

J. Am. Chem. Soc.

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123

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Identification of the Purine Nucleotides a and b as the 2'- and 3'-Phosphoribosides, Respectively¹

Cited by 30 publications

References 4 publications

Transition States of Uncatalyzed Hydrolysis and Aminolysis Reactions of a Ribosomal P-Site Substrate Determined by Kinetic Isotope Effects

Transition States of Uncatalyzed Hydrolysis and Aminolysis Reactions of a Ribosomal P-Site Substrate Determined by Kinetic Isotope Effects

On the Site of Esterification of Amino Acids to Soluble RNA^*

The Catalytic Hydrogenation of Pyrimidine Nucleosides and Nucleotides and the Isolation of their Ribose and Respective Ribose Phosphates^1,2

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Identification of the Purine Nucleotides a and b as the 2'- and 3'-Phosphoribosides, Respectively1

Cited by 30 publications

References 4 publications

Transition States of Uncatalyzed Hydrolysis and Aminolysis Reactions of a Ribosomal P-Site Substrate Determined by Kinetic Isotope Effects

Transition States of Uncatalyzed Hydrolysis and Aminolysis Reactions of a Ribosomal P-Site Substrate Determined by Kinetic Isotope Effects

On the Site of Esterification of Amino Acids to Soluble RNA*

The Catalytic Hydrogenation of Pyrimidine Nucleosides and Nucleotides and the Isolation of their Ribose and Respective Ribose Phosphates1,2

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Identification of the Purine Nucleotides a and b as the 2'- and 3'-Phosphoribosides, Respectively¹

On the Site of Esterification of Amino Acids to Soluble RNA^*

The Catalytic Hydrogenation of Pyrimidine Nucleosides and Nucleotides and the Isolation of their Ribose and Respective Ribose Phosphates^1,2