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The Binding of Nucleotides by Pancreatic Ribonuclease

I. PROTON UPTAKE AND RELEASE ASSOCIATED WITH ANION BINDING

From the ¹ From the Chemisches Institut der Universität Marburg, Marburg an der Lahn, Germany

Measurements are made of the change in the H⁺ concentration of unbuffered ribonuclease solutions caused by the addition of various competitive inhibitors at the same pH. By adjustment of the pH back to the initial value, the H⁺ taken up or released per mole of enzyme is measured.

Above pH 5.5, mixtures of RNase and 2'-cytidylate become more alkaline than the separate solutions, whereas below pH 5.5, the mixtures become more acidic. At pH 5.5, where RNase-nucleotide complexes are most stable, the H⁺ concentration remains unchanged. Just above or below this pH, small changes in the pH are noted until 1 mole of 2'-cytidylate per mole of RNase is added; thereafter the pH remains constant. A greater excess of nucleotide must be added in order to reach maximum H⁺ uptake above pH 6 or maximal H⁺ release below pH 5. The addition of a 4-fold excess of 2'-cytidylate to 1.6 x 10^-4 m RNase causes an uptake of about 1.4 H⁺ per mole of RNase at pH 7 and a release of about 1.1 H⁺ per mole of RNase at pH 4. At very high concentrations of cytidylate, the uptake in alkaline ranges approaches 2 H⁺ per mole of enzyme. Similar, but much weaker, changes in the H⁺ concentration are noted when 2'-cytidylate is added to guanidinated RNase, whereas none take place when it is added to carboxymethylhistidine-119-RNase.

Proton uptake above pH 5.5 and release below pH 5.5 occurs upon addition to RNase of 3'-uridylate and 2'-adenylate, as well as of a number of non-nucleotide inhibitors bearing a terminal phosphate group. The pH at which no H⁺ uptake or release takes place is about 5.5, regardless of the dissociation constants of the phosphate groups involved. No change in the pH is caused by addition of nucleosides or nonsubstrate nucleoside cyclic phosphates.

Addition of sulfate or trimetaphosphate, which are incapable of partial protonation, to RNase causes only a weak H⁺ uptake, which is greatest at pH 5.5. Polyglucose sulfate causes a large H⁺ uptake but also precipitates the enzyme.

These results are interpreted in terms of equilibria between various ionized forms of the binding site of the enzyme, only one of which is able to form a stable complex with the phosphate group of the inhibitor. As a result of complex formation, displacement of the equilibrium between the various ionized species would cause H⁺ uptake above, and H⁺ release below, the pH at which the binding species predominates. The two groups at the binding site are estimated to have pK values of about 5.1 and 6.1, respectively. The excess H⁺ uptake on the alkaline side of the pH of maximal binding indicates that an additional protonation of either the phosphate group or the binding site is also involved in complex formation. Interactions involving both electrostatic and hydrogen bonding between the phosphate group and the binding site appear to explain the considerable stability of RNase-nucleotide complexes.

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