Abstract
Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisincatalyzed semisynthesis starting from synthetic RNase 1‐20 peptides and S‐protein (RNase 21‐124). The lysyl residue at position 1 was replaced by alanine, whereas Lys‐7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl‐3′,5′‐adenosine and yeast RNA. When Lys‐7 was replaced by S‐methyl‐cysteine or S‐carboxamido‐methyl‐cysteine, binding of recombinant ribonuclease inhibitor (RI) from porcine liver was strongly affected. In contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A‐RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys‐1, Cys‐7‐methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol^−1^. Modifications that introduced a positive charge in position 7 (S‐aminoethyl‐ or S‐ethylpyridyl‐cysteine) led to much smaller losses. The replacement of Lys‐1 resulted in a 4‐kJ mol^−1^ loss in binding energy. S‐protein bound to R1 with K~1~ = 63.4 pM, 800‐fold weaker than RNase A. This corresponded to a 16‐kJ mol^−1^ difference in binding energy. The results show that the N‐terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys‐7 and to a smaller extent of Lys‐1 provide most of the binding energy.