Lacadena J, Mancheño J M, Martinez-Ruiz A, Martínez del Pozo A, Gasset M, Oñaderra M, Gavilanes J G
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):581-6. doi: 10.1042/bj3090581.
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.
α-肌动蛋白细胞毒素是一种细胞外真菌蛋白，它通过特异性切割28 S rRNA的一个磷酸二酯键来抑制蛋白质生物合成。基于与一些真菌核糖核酸酶的序列相似性，α-肌动蛋白的His137残基被认为参与了该蛋白的催化活性。用Gln取代该残基（α-肌动蛋白的H137Q突变变体）会消除该蛋白的核糖核酸酶活性。通过测量其对完整兔核糖体和均聚物聚（A）的作用，已在H137Q α-肌动蛋白的均一制剂中证明了这一点。H137Q α-肌动蛋白的构象与野生型蛋白高度相似，这已通过圆二色光谱和荧光光谱进行了分析。H137Q和野生型α-肌动蛋白在肽键区域均表现出相同的圆二色光谱，表明突变后二级结构水平没有变化。与野生型蛋白相比，在近紫外圆二色光谱和荧光发射光谱中仅观察到微小差异。此外，H137Q α-肌动蛋白与磷脂囊泡相互作用，产生与天然细胞毒素相同的效果。因此，我们认为His137是细胞毒素α-肌动蛋白核糖核酸酶活性位点的一部分。
