title: Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin.
abstract: alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Universidad Complutense, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Lacadena
authors.forename: A
authors.initials: A
authors.lastname: Martínez del Pozo
authors.forename: A
authors.initials: A
authors.lastname: Martínez-Ruiz
authors.forename: J M
authors.initials: JM
authors.lastname: Pérez-Cañadillas
authors.forename: M
authors.initials: M
authors.lastname: Bruix
authors.forename: J M
authors.initials: JM
authors.lastname: Mancheño
authors.forename: M
authors.initials: M
authors.lastname: Oñaderra
authors.forename: J G
authors.initials: JG
authors.lastname: Gavilanes
mesh_terms: D001230:Aspergillus / Q000737:chemistry*; D020134:Catalytic Domain; D002942:Circular Dichroism; D003603:Cytotoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004722:Endoribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D003603:Cytotoxins; D005656:Fungal Proteins; D011994:Recombinant Proteins; C014801:alpha-sarcin; D018698:Glutamic Acid; D006639:Histidine; D004722:Endoribonucleases