Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor.
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-carboxamido-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 Ki = 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.
Neumann
U
U
Friedrich Miescher-Institut, Basel, Switzerland.
Hofsteenge
J
J
D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006860:Hydrogen Bonding; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism; D013552:Swine; D013816:Thermodynamics
D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011506:Proteins; D012331:RNA, Fungal; D011994:Recombinant Proteins; C066953:ribonuclease inhibitor, porcine; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D013381:Subtilisins