Substitution of histidine-137 by glutamine abolishes the catalytic activity of the ribosome-inactivating protein alpha-sarcin.
The alpha-sarcin cytotoxin is an extracellular fungal protein that inhibits protein biosynthesis by specifically cleaving one phosphodiester bond of the 28 S rRNA. The His137 residue of alpha-sarcin is suggested to be involved in the catalytic activity of this protein, based on the observed sequence similarity with some fungal ribonucleases. Replacement of this residue by Gln (H137Q mutant variant of alpha-sarcin) abolishes the ribonuclease activity of the protein. This has been demonstrated for an homogeneous preparation of the H137Q alpha-sarcin by measuring its effect against both intact rabbit ribosomes and the homopolymer poly(A). The conformation of H137Q alpha-sarcin is highly similar to that of the wild-type protein, which has been analysed by CD and fluorescence spectroscopy. Both H137Q and wild-type alpha-sarcin exhibit identical CD spectra in the peptide-bond region, indicating that no changes at the level of the secondary structure are produced upon mutation. Only minor differences are observed in both near-UV CD and fluorescence emission spectra in comparison to those of the wild-type protein. Moreover, H137Q alpha-sarcin interacts with phospholipid vesicles, promoting the same effects as the native cytotoxin. Therefore, we propose that His137 is part of the ribonucleolytic active site of the cytotoxin alpha-sarcin.
Lacadena
J
J
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Mancheño
J M
JM
Martinez-Ruiz
A
A
Martínez del Pozo
A
A
Gasset
M
M
Oñaderra
M
M
Gavilanes
J G
JG
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000737:chemistry; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D017931:DNA Primers; D004722:Endoribonucleases; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D005656:Fungal Proteins; D010743:Phospholipids; D005973:Glutamine; C014801:alpha-sarcin; D006639:Histidine; D004722:Endoribonucleases