Binding of phosphate ligands to ribonuclease A

Anderson, David G.; Hammes, Gordon G.; Walz, Frederick G.

doi:10.1021/bi00845a004

Cited by 168 publications

(133 citation statements)

References 23 publications

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“…An interaction of the base with this residue would explain our observations and may also be a basis for an explanation of the difference spectra obtained after mixing nucleotides with the enzyme [19,21,39]. Further studies with other nucleotides (see H o l i and sorm [40]) and calorimetric measurements now in progress will certainly provide more information on the nature of the interactions between the enzyme and the bases, which obviously contribute cooperatively to the binding strength.…”

Section: Binding Of Inhibitors To the Enzymementioning

confidence: 64%

NMR‐Studies on the Structure of the Active Site of Pancreatic Ribonuclease A

Rüterjans

Witzel²

1969

European Journal of Biochemistry

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The chemical shift of the C-2 imidazole protons of the histidine residues of pancreatic ribonuclease has been studied as a function of pH in 2Hz0. Upon protonation of the imidazole ring the C-2 proton magnetic resonance signal shifts about 1.0 ppm to a lower field. Thus titration curves and approximate pK values of the histidine residues are obtained. The pK values of histidine 119 and histidine 12 of the active site are unusually low and shift by 1.0 and 0.7 units to higher values when the NaCl concentration is changed from 0.1 M to 0.4 M, whereas the pK value of histidine 105 shifts only by about 0.2 units. This indicates the presence of a positively charged group in the immediate environment of histidine 119 and histidine 12. The abnormal chemical shift of the histidine 48 C-2 proton a t low pH is explained by means of an interaction of histidine 48 with the carboxylate anion of aspartic acid 14. From an analysis of the titration curves of histidine 119 and histidine 12 the existence of an enzyme species in which both imidazoles are connected by a hydrogen bond is derived. The C-2 proton magnetic resonance signals of histidine 119 and histidine 12 shift to a lower field when inhibitors bind to the enzyme. Both histidines are then kept in a protonated form up to the pH range of 7-8. The apparent pK values are higher in the cytidine 2'-phosphate-ribonuclease complex than in the cytidine 3'-phosphateribonuclease complex. A similar shift of the histidine I19 and histidine 12 signals is observed with binding of pyrophosphate to ribonuclease indicating that only the phosphate group causes the behaviour of the histidines. Shifts of the C-6 proton magnetic resonance signal of the pyrimidine ring indicate an additional binding of the base t o an aromatic side chain of the enzyme.The mechanism for the binding process proposed in previous papers by Witzel and coworkers in which histidine 119 and lysine 41 are the binding groups of the enzyme, whereas histidine 12 takes up a proton when being released from its binding to histidine 119, is in agreement with the results reported in this communication.Upon protonation of the imidazole ring of histidine the proton magnetic resonance signal of the C-2 proton (compare Scheme 1) shifts downfield by about If it is assumed that the inhibitors bind to the histidines 119 or 12 or both then there should be characteristic changes in the pK values of these histidines. If these shifts in pK are also accompanied by linewidth changes of the signals, it should be possible to confirm some mechanistic features of the binding process already developed from kinetic studies of the reaction with substrates and inhibitors

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Section: Binding Of Inhibitors To the Enzymementioning

confidence: 64%

NMR‐Studies on the Structure of the Active Site of Pancreatic Ribonuclease A

Rüterjans

Witzel²

1969

European Journal of Biochemistry

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“…Three proteins known to possess these properties were examined, Staphylococcal nuclease (25,26), ribonuclease (27,28), and cytochrome c (29,30). As shown in Table 1 The column labeled "pI" lists the isoelectric point or isoionic point for the proteins as reported in the appropriate volumes of The Enzymes.…”

Section: Methodsmentioning

confidence: 99%

“…* Affinity chromatography using ribonuclease was done in 10 mM acetate buffer (pH 5.5) since the ligand 2'-CMP binds most strongly at this pH (28 apoflavodoxin do not bind to the affinity columns even though they each contain a remnant of the dinucleotide fold.…”

Section: Methodsmentioning

confidence: 99%

Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins.

Thompson¹,

Cass²,

Stellwagen³

1975

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

393

144

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A procedure is described to utilize blue dextran-Sepharose as an affinity chromatographic column specific for the super-secondary structure called the dinucleotide fold, which forms the binding sites for substrates and effectors on a wide range of proteins. The procedure can be used to identify proteins, either purified or in crude cellular extracts, that possess the dinucleotide fold and to significantly improve the purification procedures for those proteins that possess the fold.A sulfonated polyaromatic blue dye covalently attached to dextran, called blue dextran,O-DEXTRAN is commonly used to measure the void volume of exclusion chromatographic columns. It has been observed that several proteins that normally penetrate the internal volume of exclusion gel beads are totally excluded from the internal volume when chromatographed along with blue dextran in the presence of low, but not high, ionic strength solvents. A variety of proteins exhibit this behavior, such as pyruvate kinase (1, 2), glutathione reductase (3), and blood coagulation factors II, VII, IX, and X (4). It was concluded that blue dextran forms a complex with each of these proteins that is dissociable by salt. Subsequent to these observations, blue dextran-Sepharose affinity columns have been made and used in the final purification stages of enzymes such as phosphofructokinase (5) and lactate dehydrogenase (6).These enzymes were displaced from the affinity columns by addition of low concentrations of their nucleotide substrates to the elution solvents.We propose that the blue dextran complexes with this wide range of proteins because it is specific for a super-secondary structure called the dinucleotide fold. This structure involves about 120 amino acids that are arranged in a ,-sheet core composed of five or six parallel strands connected by ahelical intrastrand loops located above and below the ,8-sheet (7,8). The dinucleotide fold is known to form the NADbinding site in lactate (9), malate (10), and glyceraldehydephosphate (11) dehydrogenases, to form the ATP-binding site in phosphoglycerate kinase (12), and to be present in the structures of alcohol dehydrogenase (13), adenylate kinase (14,15), and phosphoglycerate mutase (16). In addition, the FMN-binding site of flavodoxin (17,18) and the aromatic specificity site in the proteolytic enzyme subtilisin (19,20) have been proposed (7,8) to contain a remnant of the dinucleotide fold termed the mononucleotide fold. Accordingly, we examined the interaction of blue dextran-Sepharose affinity columns with each of these proteins as well as with several proteins known not to possess the dinucleotide fold to determine the specificity of the affinity columns. MATERIALS AND METHODS

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“…Four aliquots, approximately 2.5 to 5 ml each, of RNase A stock solution were diluted 1:1 (v/v) with one of the four salt stock solutions (Na ϩ , K ϩ , Ca 2ϩ , or Mg 2ϩ acetate) and then buffered to the optimal 2Ј-CMP binding pH of 5.5 (Anderson et al, 1968) by dropwise addition of 50 mM acetic acid. The actual RNase concentration in each working solution was determined by quantitative UV spectrophotometry (Hewlett-Packard 8453 diode-array spectrophotometer; 277.5 nm; extinction coefficient ⑀ ϭ 9800 M Ϫ1 cm Ϫ1 ; pH 5.5).…”

Section: Methodsmentioning

confidence: 99%

Toward the Design of Ribonuclease (RNase) Inhibitors: Ion Effects on the Thermodynamics of Binding of 2′-CMP to RNase A

Spencer

Abdul²,

Schulingkamp³

et al. 2002

J Pharmacol Exp Ther

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Ribonucleases (RNases) possess a variety of biological activities and, under certain conditions, are deleterious. Hence, design of selective inhibitors has been suggested as a strategy for treating RNase-related disorders. In the present study, isothermal titration calorimetry was used to measure ion effects on binding thermodynamics of the RNase A competitive inhibitor 2Ј-CMP as a representative system. The reaction cell (37°C) contained dialyzed RNase A (0.04 -0.05 mM) in buffered solution (pH 5.5) of 50 mM Na ϩ , K ϩ , Ca 2ϩ , or Mg 2ϩ acetate, verified spectrophotometrically. Thirty-five sequential injections (4 l each, 3 min apart) were made of 2Ј-CMP (1.2 mM) in ionmatching buffer. The data were corrected for heat of dilution. There was a 1:1 interaction in each case. The estimated parameters (ϮS.D.) were: K d ϭ 4.84 Ϯ 0.29 M (Na ϩ ); 5.62 Ϯ 0.98 ). Thus, all reactions were enthalpy-driven. Despite a 5-fold difference in K d between mono-and divalent ions, the ratio of ion hydration ⌬G o to K d was constant. These data should be useful for molecular modeling and suggest that inhibitor activity will be a function of cellular conditions (normal or pathological).

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Binding of phosphate ligands to ribonuclease A

Abstract: To whom reprint requests should be directed.

Cited by 168 publications

References 23 publications

NMR‐Studies on the Structure of the Active Site of Pancreatic Ribonuclease A

NMR‐Studies on the Structure of the Active Site of Pancreatic Ribonuclease A

Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins.

Toward the Design of Ribonuclease (RNase) Inhibitors: Ion Effects on the Thermodynamics of Binding of 2′-CMP to RNase A

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