INTERACTION OF SEMISYNTHETIC VARIANTS OF RNASE-A WITH RIBONUCLEASE INHIBITOR

Derivatives of ribonuclease A (RNase A) with modifications in position s 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis start ing from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). T he lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semis ynthesis. 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-carboxamidomethyl-c ysteine, binding of recombinant ribonuclease inhibitor (RI) from porci ne 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-methy l RNase), corresponding to a decrease in binding energy of 10 kJ mol(- 1). Modifications that introduced a positive charge in position 7 (S-a minoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol(-1) loss in binding energ y. S-protein bound to RI with K-i = 63.4 pM, 800-fold weaker than RNas e 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 inte ractions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.