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, F120G, 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 parallel decrease in amino-acid volume at position 120, while the destabilization of F120W can be ascribed to an increase in DeltaS(m) accompanying an increase in DeltaH(m), showing that the size of Phe120 produces an optimum balance of conformational enthalpy and entropy for achieving the maximal structural stability. Moreover, the replacement of Phe120 affects activity. The increase in K(m) showed that the hydrophobicity and pi electron of Phe120 are important factors in substrate binding. The decrease in k(cat) was predicted to be due to positional changes of the side chains of His12 and/or His119. The positional changes were successfully detected by the rate of carboxymethylation by iodoacetate or bromoacetate, which correlated very well with decreases in activity, supporting the view that Phe120 also plays an important role in determining the position of His12 and/or His119 in order to achieve efficient catalysis.
Chatani
E
E
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Tanimizu
N
N
Ueno
H
H
Hayashi
R
R
D000085:Acetates / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics
D000085:Acetates; D007461:Iodoacetates; D010710:Phosphates; D011994:Recombinant Proteins; D010649:Phenylalanine; C016274:bromoacetate; D012259:Ribonuclease, Pancreatic