Structural and functional changes in bovine pancreatic ribonuclease A by the replacement of Phe120 with other hydrophobic residues

To clarify the specific role of Phe120 in bovine pancreatic ribonuclease A (RNase A), changes in the thermal stability and activity of F120L, F120A, F 120G, and F120W were analyzed with respect to some thermodynamic terms, i.e ., Gibbs free energy, enthalpy, and entropy. The structural destabilization of F120L, F120A, and F120G: was due to a decrease in DeltaH(m) with a para llel decrease in amino-acid volume at position 120, while the destabilizati on of F120W can be ascribed to an increase in DeltaS(m) accompanying an inc rease in DeltaH(m), showing that the size of Phe120 produces an optimum bal ance of conformational enthalpy and entropy for achieving the maximal struc tural stability. Moreover, the replacement of Phe120 affects activity. The increase in K-m showed that the hydrophobicity and ii electron of Phe120 ar e important factors in substrate binding. The decrease in k(cat) was predic ted to be due to positional changes of the side chains of His12 and/or His1 19. The positional changes were successfully detected by the rate of carbox ymethylation by iodoacetate or bromoacetate, which correlated very well wit h decreases in activity, supporting the view that Phe120 also plays an impo rtant role in determining the position of His12 and/or His119 in order to a chieve efficient catalysis.