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The reversible unfolding and folding of staphylococcal nuclease in the acid transition has been studied by 220 MHz proton magnetic resonance spectroscopy. The values of area, line-width, and chemical shift of each of the imidazole C2 proton resonances of the four histidine residues have been measured in this transition. The change of areas of three histidine resonances and the change of fluorescence of the single tryptophan residue, as a function of pH, appear to follow a single equilibrium. In contrast, a fourth histidine resonance follows a biphasic transition. These findings indicate that local conformational changes can be detected by magnetic resonance spectroscopy in the cooperative transition of the overall structure.

The possible mechanisms of action of bovine pancreatic ribonuclease are discussed in the light of the detailed knowledge of the geometry of the active site that has been derived from studies of inhibitor binding by X-ray diffraction and nuclear magnetic resonance. When combined with a knowledge of the mechanism of phosphate ester hydrolysis, this information imposes severe geometric constraints on possible mechanisms of action of the enzyme. Two types of mechanism can be distinguished, the linear and the pseudorotation. The linear mechanism includes a catalytic role for both histidine residues at the active site and does not involve pseudorotation of the intermediate. In contrast, in the pseudorotation mechanism one histidine residue performs all the catalytic functions, while the other serves only to bind the phosphate anion; this necessarily involves pseudorotation of the intermediate and specific protonation of the leaving group by the enzyme. The mode of binding of the product of the reaction, cytidine-3'-monophosphate, has been elucidated by X-ray diffraction and nuclear magnetic resonance. If the substrate binds in an analogous way, only the linear mechanism is possible. This mechanism is described in detail.