Nucleoside-amino acid conjugates have been employed to inhibit the ribonucleolytic activity of ribonuclease A (RNase A) and affect the protonation/deprotonation equilibrium of its active site histidine residues. Agarose gel and precipitation assays indicate inhibition of RNase A activity by these molecules with a possible role of the polar side chains of the amino acids in RNase A inhibition. Kinetic experiments demonstrated that the mode of inhibition is competitive in nature with inhibition constants (K(i)) in the micromolar range. The nucleoside-serine conjugate occupies the active site of RNase A and preferential perturbs the pK(a) value of His-119 by its 'free amino group' as found from (1)H NMR studies. Docking studies revealed that the free amino groups of the most active compounds are within hydrogen bonding distance of His-119 in inhibitor-RNase A complexes.