NUCLEAR MAGNETIC RESONANCE STUDIES OF STRUCTURE AND BINDING SITES OF ENZYMES .8. INHIBITOR BINDING TO RIBONUCLEASE

Changes are observed in the aromatic region of the nuclear magnetic spectrum of bovine pancreatic ribonuclease A on addition of the inhibitors cytidine-2′-monophosphate and cytidine-5′-monophosphate. These are described and compared with those previously observed in the presence of cytidine-3′-monophosphate. Addition of these inhibitors causes the pK values of histidine 12 and histidine 119 to increase and their C(2)-H peaks to shift. The magnitudes of the peak shift and pK increase of histidine 119 vary with the position of the phosphate group and can be correlated with the binding strength of the inhibitor (2′ > 3′ > 5′). Both of these histidine residues are positively charged in the complexes with cytidine-2′-monophosphate and cytidine-3′-monophosphate. The pK value and chemical shift of the C(2)-H peak of histidine 105 are unaffected by inhibitor binding. The histidine 48 peak shifts slightly in the presence of inhibitor, indicating that inhibitor binding effects the conformational equilibrium of the enzyme. The nature of this equilibrium is discussed. In order to specify the interactions of the different chemical groups of the mononucleotide inhibitors with ribonuclease, the effects of the binding of the inhibitors cytidine and phosphate on the nuclear magnetic resonance spectrum of the enzyme were also observed. Further information concerning the environment of the inhibitor in the active site was obtained from the shifts of the C(6)-H, C(5)-H and C(1′)-H peaks of the nucleotide and nucleoside inhibitors. Models of the structures of the ribonuclease-mononucleotide complexes are postulated which are consistent with the nuclear magnetic resonance data and with the results of studies of the structure of ribonuclease by other methods. © 1969.