1H Nmr study on the binding of CMP inhibitors to RNase A. III. Chemical exchange and relaxation effects

Abstract

A new dynamic 1 H nmr method has been developed and applied to the binding of 3′- and 5′-cytidine monophosphate (CMP) to Ribonuclease A. The study has provided information on the rates of chemical exchange and the degree of inhibitor immobilization at the active site. Evidence is presented which indicates that exchange-broadening effects on the line widths result from a slow pH-dependent conformational change of the enzyme inhibitor complex. At pH 4.5–5.5 and 24° 1/τ∼150–250 sec −1 for the CMP inhibitors with the exchange rate increasing with increasing pH. The rotational correlation time for the pyrimidine ring of the complex is 4.4 × 10 −9 see and for the ribose ring of the complex is 1.2 × 10 −8 sec. These results indicate that the ribose ring is rigidly bound to the enzyme while there remains some residual degree of rotational freedom about the glycosidic bond.