A STUDY OF CONFORMATIONAL PROPERTIES OF BOVINE PANCREATIC RIBONUCLEASE A BY ELECTRON PARAMAGNETIC RESONANCE

A technique is reported for spin labeling bovine pancreatic ribonuclease A and using electron paramagnetic resonance spectroscopy to study its conformational properties. Bromo acid nitroxide and bromo amide nitroxide spin labels react with the histidine residues at the active site of the enzyme, and to a much lesser extent with lysine residues on the exterior of the protein molecule. A five-membered maleimide nitroxide spin label reacts only with these lysine residues, whereas a six-membered maleimide nitroxide label reacts with lysine-41 at the active site. Transition temperature measurements indicate that the spin-labeled derivatives are structurally very similar to native ribonuclease (RNase) A. Evidence for the binding of substrate ribonucleic acid (RNA) and synthetic polyribonucleotides to spin-labeled RNase A, RNase S, and RNase S protein is presented; these data indicate that although enzymic action has been suppressed by labeling, the sites involved in recognition and binding of substrates are still intact and operational. Direct observation of the response of spin labels in the region of the active site when various perturbations are applied to RNase yields results in accord with current views of the molecular structure. However, perturbations of the tyrosine residues do not always correlate with perturbations of the active site. RNase S appears to have an active site environment very similar to that of RNase A. The temperature dependence of the electron paramagnetic resonance spectra yields activation energies related to the expansion of the active site; the magnitudes of these energies indicate that only a small number of bonds must be disrupted to destroy the three-dimensional structure necessary for enzymic activity. © 1968, American Chemical Society. All rights reserved.