STRUCTURAL INVESTIGATION OF CATALYTICALLY MODIFIED F120L AND F120Y SEMISYNTHETIC RIBONUCLEASES

The structures of two catalytically modified semisynthetic RNases obta ined by replacing phenylalanine 120 with leucine and tyrosine have bee n determined and refined at a resolution of 2.0 Angstrom (R = 0.161 an d 0.184, respectively). These structures have been compared with the r efined 1.8-Angstrom structure (R = 0.204) of the fully active phenylal anine-containing enzyme (Martin PD, Doscher MS, Edwards BFP, 1987, J B iol Chem 262:15930-15938) and with the catalytically defective D121A ( 2.0 Angstrom, R = 0.172) and D121N (2.0 Angstrom, R = 0.186) analogs ( deMel VSJ, Martin PD, Doscher MS, Edwards BFP, 1992, J Biol Chem 267:2 47-256). The movement away from the active site of the loop containing residues 65-72 is seen in ah three catalytically defective analogs - F120L, D121A, and D121N - but not in the fully active (or hyperactive) F120Y. The insertion of the phenolic hydroxyl of Tyr 120 into a hydro gen-bonding network involving the hydroxyl group of Ser 123 and a wate r molecule in F120Y is the likely basis for the hyperactivity toward u ridine 2',3'-cyclic phosphate previously found for this analog (Hodges RS, Merrifield RB, 1974, Int J Pept Protein Res 6:397-405) as well as the threefold increase in K-M for cytidine 2',3'-cyclic phosphate fou nd for this analog by ourselves.