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Protein Science, Vol 3, Issue 2 248-256, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor

U. NEUMANN and J. HOFSTEENGE
Friedrich Miescher-Institut, CH-4002 Basel, Switzerland

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed 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-carboxamidomethyl-cyste ine, 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 RI with K(i) = 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.
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