title: 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 2.0 A (R = 0.161 and 0.184, respectively). These structures have been compared with the refined 1.8-A 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 A, R = 0.172) and D121N (2.0 A, 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.
authors.affiliation: Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: V S
authors.initials: VS
authors.lastname: deMel
authors.forename: M S
authors.initials: MS
authors.lastname: Doscher
authors.forename: M A
authors.initials: MA
authors.lastname: Glinn
authors.forename: P D
authors.initials: PD
authors.lastname: Martin
authors.forename: M L
authors.initials: ML
authors.lastname: Ram
authors.forename: B F
authors.initials: BF
authors.lastname: Edwards
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005973:Glutamine; D006639:Histidine; D006860:Hydrogen Bonding; D008239:Lysine; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D012260:Ribonucleases / Q000138:chemical synthesis / Q000737:chemistry*; D012984:Software; D014867:Water / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D014867:Water; D005973:Glutamine; D001224:Aspartic Acid; D006639:Histidine; D012260:Ribonucleases; D008239:Lysine