Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases

• Published in: Protein science Vol. 3; no. 1; pp. 39 - 50
• Main Authors: Demel, V. Srini J., Doscher, Marilynn S., Glinn, Michele A., Martin, Philip D., Ram, Michal L., Edwards, Brian F.P.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01-01-1994
• Subjects: Amino Acid Sequence; Aspartic Acid; Binding Sites; comparison of active site structure; Crystallography, X-Ray; Glutamine; Histidine; Hydrogen Bonding; loop conformational change; Lysine; modulation of hydrogen bonding; Molecular Sequence Data; Molecular Structure; mutants; Peptide Fragments - chemistry; Protein Conformation; protein semisynthesis; Ribonucleases - chemical synthesis; Ribonucleases - chemistry; semisynthetic RNases; Software; Water - chemistry; X‐ray structure
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560030106

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 2.0 Å (R = 0.161 and 0.184, respectively). These structures have been compared with the refined 1.8‐Å structure (R = 0.204) of the fully active phenylalanine‐containing enzyme (Martin PD, Doscher MS, Edwards BFP, 1987, J Biol Chem 262:15930‐15938) and with the catalytically defective D121A (2.0 Å, R = 0.172) and D121N (2.0 Å, R = 0.186) analogs (deMel VSJ, Martin PD, Doscher MS, Edwards BFP, 1992, J Biol Chem 267:247‐256). The movement away from the active site of the loop containing residues 65‐72 is seen in all 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 hydrogen‐bonding network involving the hydroxyl group of Ser 123 and a water molecule in F120Y is the likely basis for the hyperactivity toward uridine 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 KM for cytidine 2′,3′‐cyclic phosphate found for this analog by ourselves.