STUDIES ON RIBONUCLEASE S: THE ROLE OF LYSINE‐7 FOR ACTIVATION OF S‐PROTEIN*

In order to describe further the role of the invariant amino acid residue lysine-7 in the RNase S' system, potential catalytic activities of des-Lys7-[Orn10]-, [Ala7, Orn10]-, [Lys4, Ala7, Orn1.0]-S-peptide and corresponding guanidinated derivatives were studied. New information was obtained about S-protein binding capacities of the analogs relative to that of unmodified S-peptide, and about conformation of the corresponding RNase S' analogs by studies on competitive inhibition, difference spectroscopy, difference circular dichroism and thermal transition. Peptides modified in position 7 were no weaker in strength in peptide-protein noncovalent interaction in the presence of substrate. In the absence of substrate the variously modified S-peptide analogs were differentiated in their S-protein affinity. Conformational changes in the geometry of the active site of the reconstituted enzymes, if present, must be smaller than the detection limits of the techniques used. There is strong implication that the change in catalytic efficiency has to be attributed to an alteration or to the position of the charge and that lysine-7 in the natural enzyme is involved in stabilizing the transition state intermediates during decomposition of the substrate. Further substantiation was provided by substrate inhibition studies as well as 3-CMP binding measurements, since 3-CMP binding affinity of RNase S' modified in position 7 was found to be significantly lower than that of RNase S' itself.