Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin.
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.
Lacadena
J
J
Departamento de Bioquímica y Biología Molecular, Universidad Complutense, Madrid, Spain.
Martínez del Pozo
A
A
Martínez-Ruiz
A
A
Pérez-Cañadillas
J M
JM
Bruix
M
M
Mancheño
J M
JM
Oñaderra
M
M
Gavilanes
J G
JG
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
D003603:Cytotoxins; D005656:Fungal Proteins; D011994:Recombinant Proteins; C014801:alpha-sarcin; D018698:Glutamic Acid; D006639:Histidine; D004722:Endoribonucleases