Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases

Abstract

The structures of two catalytically modified semisynthetic RNases obtained by replacing phenylalanine 120 with leucine and tyrosine have been determined and refined at a resolution of 2.OA ( R = 0.161 and 0.184, respectively). These structures have been compared with the refined 1.

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

“…Here, it should be noted that the highest deviation was only 1.3 Å (F120G), which results in a decrease in 93.3% of k cat for CpA. Although the scale of the deviation was much smaller than expected, the similar correlation is also observed in the other sets of mutant en-zymes, that is, D121N and D121A and in semisynthetically prepared F120Y and F120L RNase A (it should be noted that the pK a of His12 and 119 of these mutant enzymes are the same as that of the wild-type enzyme; de Mel et al 1994a;Schultz et al 1998b), all of the deviations of which were <1.0 Å (Table 3). The obvious correlations shown in Table 3 indicate the possibility that a 1-Å deviation might drastically reduce activity.…”

“…A decrease in RNase A activity had been ascribed to the conformational change of the 65-72 loop region, according to the experimental results used by semisynthetic F120L, D121N, and D121A RNase A (de Mel et al 1992(de Mel et al , 1994a. .…”

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 Å resolution

“…Here, it should be noted that the highest deviation was only 1.3 Å (F120G), which results in a decrease in 93.3% of k cat for CpA. Although the scale of the deviation was much smaller than expected, the similar correlation is also observed in the other sets of mutant enzymes, that is, D121N and D121A and in semisynthetically prepared F120Y and F120L RNase A (it should be noted that the pK a of His12 and 119 of these mutant enzymes are the same as that of the wild‐type enzyme; de Mel et al 1994a; Schultz et al 1998b), all of the deviations of which were <1.0 Å (Table 3). The obvious correlations shown in Table 3 indicate the possibility that a 1‐Å deviation might drastically reduce activity.…”

“…A decrease in RNase A activity had been ascribed to the conformational change of the 65–72 loop region, according to the experimental results used by semisynthetic F120L, D121N, and D121A RNase A (de Mel et al 1992, 1994a). This also appears to be supported by the present results, in that the loop region of the three Phe120 mutant RNase A appears to move away from the active site.…”

Ribonuclease A

“…These studies were successful in illuminating molecular aspects of enzymatic catalysis, protein−protein interactions, and protein−nucleic acid interactions, and were the harbinger of current work on proteins containing variant or nonnatural amino acid residues. Work on the structure and function of another semisynthetic ribonuclease, RNase-(1−118)·(111−124), , has also made significant contributions. ,,,,− Recently, the RNase S system has spawned or at least facilitated the development of many innovative technologies.…”