On the dinitrophenylation of bovine pancreatic ribonuclease A kinetics of the reaction in water and 8M urea

Murdock, Archie L.; Grist, Ken L.; Hirs, C.H.W.

doi:10.1016/0003-9861(66)90350-x

Cited by 105 publications

(35 citation statements)

References 10 publications

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“…The values of KIM and kc,, for F120Y and cytidine 2',3'-cyclic phosphate were determined at pH 6.00 and 25 "C using a spectrophotometric assay based closely on that of Murdock et al (1966), which utilizes the increase in absorbance in the vicinity of 286 nm that occurs upon conversion to the product, 3"cytidylate (Sasaki et al, 1985). The integrity of substrate solutions was checked by measurement at pH 7.00 of the ratio of the absorbance at 265 nm to that at 286 nm; a value lower than 2.94 indicated that hydrolysis to 3"cytidylate had occurred (Crook et al, 1960).…”

Section: Methodsmentioning

confidence: 99%

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases

et al. 1994

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Section: Methodsmentioning

confidence: 99%

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases

et al. 1994

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“…The high reactivity of the corresponding RNase A residue-Lys-41-withi modification reagents is probably due in part to its abnormally low pKa value of -8.8 (28). This pKa is thought to reflect the presence of neighboring positive charges, such as Arg-39 (29) and Treatment with arginine reagents also inactivates angiogenin, indeed much faster than RNase A (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Chemical modifications were performed by standard procedures as described below using [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] t.M angiogenin.…”

Section: Methodsmentioning

confidence: 99%

Ribonucleolytic activity of angiogenin: essential histidine, lysine, and arginine residues.

Shapiro

Weremowicz²,

Riordan³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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The homology of angiogenin and pancreatic RNase A provides a compelling reason to systematically compare the characteristics of the two proteins using the chemical modification approaches that proved essential to understanding the action of RNase. Reagents specific for histidine, lysine, and arginine markedly decrease the ribonucleolytic activity of angiogenin, much as has been observed for RNase A. Activity is abolished by reduction of the disulfide bonds and is restored by reoxidation. Methionine, tyrosine, and carboxyl group reagents have no significant effect. From the point of view of reactivity, the histidine and lysine residues in angiogenin are severalfold less susceptible to modification than those in RNase A. Arginine reagents, on the other hand, inactivate angiogenin considerably faster than RNase A. Considering specificity, bromoacetate inactivates angiogenin at pH 5.5 by modifying 1.5 histidines, but lysine and arginine reagents are less specific. Thus, 3.8 and 6.3 residues, respectively, are modified by 1-fluoro-2,4-dinitrobenzene and by formaldehyde plus cyanoborohydride, under conditions where activity decreases by w80% in both cases. With phenylglyoxal, 6.7 arginnes are lost when there is 92% inactivation. Poly(G) prevents inactivation by lysine and arginine reagents, and phosphate protects against the effects of lysine modification. Thus, the functional consequences of these modifications likely reflect the loss of critical residues rather than general conformational effects.

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“…A picture for the role of Lys 41 in catalysis has developed over the last 30 years. The importance of Lys 41 was discovered by chemical modification studies (Hirs et al, 1965;Murdock et al, 1966). Subsequent structural analyses led to the proposal that Lys 41 stabilizes selectively the chemical transition state for RNA cleavage (Alber et al, 1983 creases the catalytic activity of RNase A by 20-fold and the T, by 20 "C (Laity et al, 1993 (Eftink & Biltonen, 1987).…”

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confidence: 99%

Contribution of a tyrosine side chain to ribonuclease A catalysis and stability–Contribution of Tyr 97 to RNase A catalysis and stability

et al. 1996

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An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of kc,, being perturbed more significantly than that of K,,,. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidy1ic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and l l .7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr + Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond.

show abstract

On the dinitrophenylation of bovine pancreatic ribonuclease A kinetics of the reaction in water and 8M urea

Cited by 105 publications

References 10 publications

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases

Ribonucleolytic activity of angiogenin: essential histidine, lysine, and arginine residues.

Contribution of a tyrosine side chain to ribonuclease A catalysis and stability–Contribution of Tyr 97 to RNase A catalysis and stability

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