title: 15N- and 1H-NMR investigations of the active-site amino acids in semisynthetic RNase S' and RNase A.
abstract: Extensive 15N-NMR investigations of active-site amino acids were made possible by the solid-phase synthesis of the N-terminal pentadecapeptide of RNase A with selectively 15N-enriched amino acids. On complexation with S-protein a fully active RNase S' complex was obtained. The 15N resonances of the side chains of lysine-7 (N epsilon), glutamine-11 (N gamma), and histidine-12 (N pi, tau) were studied in the free synthetic peptide, in the RNase S' complex and in the nucleotide complexes RNase S' with 2'CMP, 3'CMP, and 5'AMP. The analysis of the 15N-1H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide-protein interaction, the side chains of Lys-7 and Gln-11 do not contribute to this interaction. In the nucleotide complexes the resonances of His-12 and Gln-11 are shifted downfield. In the 2'CMP complex a doublet for the N tau signal of His-12 indicates a stable H bond between this nitrogen and the phosphate group of nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln-11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys-7 signal is line broadening due to reduced proton exchange. For comparison with the 15N-NMR titration curves of His-12 in RNase S' the 1H-NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the 15N and the 1H titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active-site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the 15N resonances, show a small inflection in the pH range 5.8-6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active-site histidines by a partial H-bond formation between the imidazole groups.
authors.affiliation: Institut für Biophysikalische Chemie, J.W. Goethe-Universität, Frankfurt, Federal Republic of Germany.
authors.forename: H
authors.initials: H
authors.lastname: Knoblauch
authors.forename: H
authors.initials: H
authors.lastname: Rüterjans
authors.forename: W
authors.initials: W
authors.lastname: Bloemhoff
authors.forename: K E
authors.initials: KE
authors.lastname: Kerling
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D001665:Binding Sites; D004735:Energy Transfer; D006639:Histidine / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D010455:Peptides / Q000032:analysis; D011522:Protons; D012259:Ribonuclease, Pancreatic / Q000032:analysis*; D012260:Ribonucleases / Q000032:analysis*; D012265:Ribonucleotides / Q000032:analysis
chemical_list: D000596:Amino Acids; D010455:Peptides; D011522:Protons; D012265:Ribonucleotides; D006639:Histidine; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C022542:ribonuclease S; D008239:Lysine