| Abstract: | 3-N-Carboxymethyl-His-12 and 1-N-carboxymethyl-His-119-RNase A bind to the naturally occurring RNase inhibitor, isolated from human placenta, 1.3 and 3.6 times, respectively, more strongly than does native RNase A. Near-ultraviolet circular dichroism measurements indicate that the conformational change which occurs upon carboxymethylation of either of the active site histidine residues appears different from that which the protein undergoes upon binding of substrate of a substrate analogue. Specific carboxymethylation of Lys-41 of RNase A decreased the strength of the interaction between the enzyme and the RNase inhibitor to about 12% of the initial value. The near-UV CD spectra of Cm-Lys-41-RNase A and of acetimidyl-RNase A (9.3 lysines modified) and carbamylated RNase A (3.0 lysines modified), which also have weaker interactions with RNase inhibitor of 25% and 10%, respectively, show a negative theta]MRW identical to that of native RNase A at 275 nm but are altered in the positive theta]MRW at 240 nm. The CD measurements suggest that one or more tyrosine residues of RNase A may be involved in the interaction with inhibitor. The effects of pH and salt concentration suggest that a major part of the protein-protein interaction is probably through nonpolar forces. The strengths of interactions between the inhibitor and pancreatic RNases from several species were very similar. Since Tyr-92 is the only tyrosine residue retained in all of the species studied, this residue may have a key role in the nonpolar interaction. The data presented herein suggest that the interaction between RNase A and the inhibitor involves the positively charged epsilon-NH2 group of Lys-41 of RNase A. This interaction could result in the inactivation of the enzyme. |
