Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 A resolution

Abstract

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants--F120A, F120G, and F120W--were analyzed up to a 1.4 A resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild-type RNase A, except for the C-terminal region of F120G with a high B-factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild-type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild-type RNase A, and those of F120W and F120G appeared to be "inside out" compared with that of wild-type RNase A. Only approximately 1 A change in the distance between N(epsilon2) of His12 and N(delta1) of His119 causes a drastic decrease in k(cat), indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.

Fig. 1.

Superimposition of the backbone structures of wild-type and mutant RNase A. This figure was drawn with the MOLSCRIPT program (Kraulis 1991). Yellow, red, green, and cyan are wild-type, F120A, F120G, and F120W enzymes, respectively. Loops 1, 2, 3, and 4 represent the loop regions of 15–25, 34–41, 65–72, and 87–97, respectively.

Fig. 2.

B-factor of wild-type and mutant RNase A. Loops 1, 2, 3, and 4 correspond to those in Fig. 1 ▶. Atoms of the side-chain at 120 are omitted from the plots. (Inset) An enlargement of the C-terminal regions. Wild-type, F120A, F120G, and F120W RNase A are drawn with black, red, green, and blue, respectively.

Fig. 3.

Stereo view of the superimposed structures of the active site in the wild-type and mutant RNase A. This figure was drawn with the MOLSCRIPT program (Kraulis 1991). Backbone structure of wild-type RNase A is drawn in the form of a white tube. Ball-and-stick models show the coordinates of Gln11, His12, Lys41, Thr45, Asn71, Asp83, Glu111, His119, and amino acid residue at the position 120. Yellow, red, green, and cyan are wild-type, F120A, F120G, and F120W RNase A, respectively.

Fig. 4.

Superimposition of His119 side-chains of wild-type and mutant RNase A. Yellow, red, green, cyan, and white are wild-type (A position), F120A (B position), F120G (B position), F120W (A position), and wild-type (B position; PDB code 1RPH) enzymes, respectively. Nitrogen and oxygen atoms in His119 and Asp121 are colored by blue and red, respectively. A water molecule observed near the His119 of F120G RNase A is also shown in green. Dashed lines show hydrogen bonds connected with the His119 side-chains.

Fig. 5.

Correlation of ΔΔH in thermal denaturation with ΔASA_partial (the change in the accessible surface area of the pocket to which amino acid residue at the position 120 belongs; a) and with ΔASA_total (b). The lines were obtained by the least-squares method with correlation coefficients of 0.922 and 0.975, respectively.

Fig. 6.

Correlation of ΔASA_total (change in the total accessible surface area; see Table 2) with ΔASA_{amino acid at 120}, which was calculated by ASA_{substituting amino acid at 120} − ASA_{phenylalanine} using the results by Miller et al. (1987) The line is obtained by the least-squares method with a correlation coefficient of 0.99.