Modification of Bovine Pancreatic Ribonuclease A with 4-Sulfonyloxy-2-nitrofluorobenzene

The kinetics of the reaction of various amino groups in RNase A with 4-sulfonyloxy-2-nitrofluorobenzene (potassium salt) was studied as a function of pH and temperature. Substitution at lysine-41 and inactivation of the enzyme proceed at identical rates (pH-independent second order rate constants at 28° are 0.43 ± 0.04 and 0.46 ± 0.06 M −1·min−1, respectively). The dissociation constant of the e-amino group of lysine-41 measured from the pH dependence was anomalously low, 7.7 ± 0.4 × 10−10 M (pKapp = 9.11) and 6.9 ± 0.3 × 10−10 M (pKapp = 9.16) for substitution and inactivation, respectively. The fact that the anomalous behavior of lysine-41 is observed at least as far as pH 10 reveals that an arginine side chain interacts with the amino group of this residue. Moreover, the kinetic behavior in the region above pH 10 is compatible with the view that the protein undergoes a conformational change which disrupts the configuration responsible for the anomalous behavior of lysine-41. The average value of the dissociation constant for the nine normal e-amino groups in RNase A from the kinetic analysis is 7.4 ± 0.9 × 10−11 M (pKapp = 10.1), in agreement with titration data. The pH-independent second order rate constant was found to be 0.93 ± 0.07 M −1·min−1. The corresponding quantities for the α-amino group in RNase A were 7.2 ± 0.1 × 10−9 M (pKapp = 8.14) and 0.057 ± 0.009 M −1·min−1, respectively. A linear relationship between pKapp and the pH-independent second order rate constant was observed to apply for the kinetically distinguishable amino groups. This relationship was used to estimate indirectly the enthalpy of ionization of the e-amino group at position 41. With the aid of this quantity, the pH-independent second order rate constants for the reaction at lysine-41 at each of six temperatures between 3.6 and 65° were determined. Transition state analysis revealed normal behavior as far as the region in which the protein enters its thermal transition above 50°. The rate constant found at 65° was of a magnitude consistent with normalization of the pKapp of lysine-41.