title: Characterization of Ustilago Ribonuclease U2. Effects of chemical modification at glutamic acid-61 and cystine-1 and of organic solvents on the enzymatic activity.
abstract: RNase U2 was purified and crystallized from the enriched culture medium (ammonium sulfate-urea-corn meal) of Ustilago sphaerogena and its characteristics were investigated. Chemical modification of RNase U2 was conducted with monoiodoacetic acid to carboxymethylate Glu-61 and with 2-methoxy-5-nitrotropone to nitrotroponylate the amino terminal residue. The amino terminal residue was modified reversibly by this reagent. Comparison of the 2'-AMP binding in the modified enzyme and the native one showed that Glu-61 is essential for the formation of the enzyme-substrate complex, while the amino terminal residue plays no important role in the enzymatic activity. The enzymatic activity and the structure of RNase U2 in aqueous organic solution were also investigated. The affinity of the enzyme for 2'-AMP, the inactivation by monoiodoacetic acid and the fluorescence intensity were examined. The profiles of the changes in the properties of the enzyme protein were consistent with those in the enzymatic activity. Fluorescence studies of the enzyme suggest that the tryptophan residue is closely related to the activity.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001487:Basidiomycota / Q000378:metabolism*; D003460:Crystallization; D003553:Cystine; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism; D005971:Glutamates; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014326:Tropanes / Q000494:pharmacology; D014588:Ustilago / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005971:Glutamates; D007461:Iodoacetates; D012997:Solvents; D014326:Tropanes; D003553:Cystine; D004720:Endonucleases; D012260:Ribonucleases