Calorimetric studies of protein-inhibitor interaction. I. Binding of 3'-cytidine monophosphate to ribonuclease A at pH 5.5

The enthalpy change (delta H degrees ') associated with the binding of Mg2+ to the sarcoplasmic reticulum calcium adenosine 5'-triphosphatase [(Ca2+)ATPase] is -76 kcal/mol. The affinity constant for Mg2+ obtained from calorimetric measurements agrees with the Km value for Mg2+ in the phosphorylation of the enzyme by inorganic phosphate (Pi). The delta H degrees ' of binding of Pi to the enzyme is -23.5 kcal/mol, and the affinity constant for Pi obtained from the calorimetry also agrees with the Km value for Pi in the phosphorylation reaction. delta H degrees ' of Mg2+ binding is reduced to -35 kcal/mol in the presence of either 20 mM Pi or 1.2 mM Ca2+ without a significant change in the affinity of the enzyme for Mg2+. delta H degrees ' of Pi binding to the enzyme drops to -8.5 kcal/mol in the presence of 10 mM Mg2+ without a significant change in the affinity of the enzyme for Pi. On the other hand, the presence of Ca2+ does not affect the delta H degrees ' for the binding of the substrate analogue 5'-adenylyl beta,gamma-imidodiphosphate [App(NH)p], and the presence of this analogue does not affect the delta H degrees ' for Ca2+ binding. The results suggest a model in which a conformational change, largely controlled by Mg2+ binding to the enzyme, leads to the formation of the covalent phosphoprotein intermediate.

Section: Resultssupporting

confidence: 59%

Calorimetric studies of ligand-induced modulation of calcium adenosine 5'-triphosphatase from sarcoplasmic reticulum

Epstein¹,

Kuriki²,

Biltonen³

et al. 1980

“…For example, Velick et al (1971) obtained values of AG = -7.3 kcal/mol, AH = -12.4 kcal/mol, AS = -16.8 eu, and ACp = -517 cal/(deg mol) for the binding of N A D + to yeast glyceraldehyde-3-phosphate dehydrogenase at 25" and pH 7.3, while, in an ultracentrifugation study of the binding of ATP to methemoglobin, Janig et al (1970) obtained values of AG = -2.67 kcal/mol, AH = -6.85 kcal/mol, and A S = -14.4 eu at 17" and pH 7.2. Finally, the binding of 3'-CMP to RNase A has been examined calorimetrically by Bolen et al who obtained values of AG = -5.1 1 kcal/mol, AH = -9.2 kcal/mol, and A S = -14.0 eu in 0.5 M NaOAc (pH 5.5) at 25" (Bolen et al, 1971).…”

Section: Discussionmentioning

confidence: 99%

Calorimetric analysis of aspartate transcarbamylase from Escherichia coli. Binding of cytosine 5'-triphosphate and adenosine 5'-triphosphate

Nm¹,

Friedland²,

Niekamp³

1975

The binding of CTP and ATP to aspartate transcarbamylase at pH 7.8 and 8.5 at 25 degrees has been investigated by equilibrium dialysis and flow microcalorimetry. The binding isotherms for CTP at both pH 7.8 and 8.5 and ATP AT PH 8.5 can be fit by a model which assumes three tight, three moderately tight, and six weak binding sites. The binding isotherms for ATP at pH 7.8 are best fit by a model which assumes six tight and six weaker sites. Both finite differenceH binding and finite differenceS binding are negative for both nucleotides at both pH values, so that the binding is enthalpy driven. For both nucleotides, finite differenceH is the same for the first two classes of binding sites, implying that the difference in the dissociation constants of these two classes of sites is the result of entropic effects. Direct pH measurements and calorimetric measurements in two buffers with very different heats of ionization (Tris and Hepes) indicate that the binding of both nucleotides is accompanied by the binding of protons. In the pH range 6.7-8.4, the number of moles of protons bound per mole of nucleotide increases as the pH decreases.

“…Ribonuclease A (lyophilized and phosphate free), purchased from Worthington Biochemical Corporation, Freehold, N.J., was used without further purification. The solutions were prepared as described by Bolen et al (1971) and the protein concentrations determined by optical density measurement at 277.5 nm, assuming a molar extinction coefficient of 9800 cm"1 mol-1 at pH 7.6 (Sela and Anfinsen, 1957). The concentration of enzyme varied from 2 to 10 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

Calorimetric and potentiometric characterization of the ionization behavior of ribonuclease A and its complex with 3'-cytosine monophoshate

Flögel¹,

Biltonen²

1975

The proton association behavior of ribonuclease A and its complex with 3'-cytosine monophosphate has been thermodynamically characterized in the pH range 4--8 at 25 degrees, mu = 0.05. Calorimetric and potentiometric titration data have been used to estimate the apparent pK values and enthalpy values for protonation of the four histidine residues of the protein, deltaHp. In the free enzyme the pK values were deduced to be 5.0, 5.8, 6.6, and 6.7 and deltaHp deduced to be -6.5, -6.5, -6.5, and -24 kcal/mol for residues 119, 12, 105, and 48, respectively. For the nucleotide-enzyme complex it was concluded that the apparent pK values of residues 119, 12, and 48 increased to an average value of about 7.2, the deltaHp values remaining constant for all histidine groups except 48. It was also concluded that only the dianionic phosphate form of the nucleotide inhibitor is bound to the enzyme in this pH range. These results are consistent with a thermodynamic model for the binding reaction in which inhibitor-enzyme association is coupled to the ionization of three imidazole residues (12, 119, and 48) and the interaction between the negative phosphate moiety of the inhibitor and the positively charged residues 12 and 119 is purely electrostatic. However, the "interaction" with residue 48 probably involves a conformational rearrangement of the macromolecule.