Substitution of histidine-137 by glutamine abolishes the catalytic activity of the ribosome-inactivating protein alpha-sarcin

• Published in: Biochemical journal Vol. 309; no. 2; pp. 581 - 586
• Main Authors: Lacadena, J, Mancheno, J.M, Martinez-Ruiz, A, Martinez del Pozo, A, Gasset, M, Onaderra, M, Gavilanes, J.G
• Format: Journal Article
• Language: English
• Published: England 15.07.1995
• Subjects: Amino Acid Sequence; amino acid substitution; Aspergillus; Aspergillus - chemistry; aspergillus giganteus; Base Sequence; binding sites; Catalysis; Chromatography, High Pressure Liquid; Circular Dichroism; cytotoxic compounds; DNA Primers; Endoribonucleases; fluorescence; Fungal Proteins - chemistry; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; glutamine; Glutamine - chemistry; histidine; Histidine - chemistry; inactivation; inhibition; interactions; Molecular Sequence Data; mutation; Phospholipids - metabolism; protein synthesis; ribosomes; Spectrophotometry, Ultraviolet; toxins
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3090581

The alpha-sarcin cytotoxin is an extracellular fungal protein that inhibits protein biosynthesis by specifically cleaving one phospho-diester 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-arcin 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 Hisl37 is part of the ribonucleolytic active site of the cytotoxin alpha-sarcin.