Tyr-48, a conserved residue in ribotoxins, is involved in the RNA-degrading activity of alpha-sarcin.
Residue Tyr-48 in alpha-sarcin is conserved not only within the ribotoxin family, but also within the larger group of extracellular fungal ribonucleases, best represented by RNase T1. A mutant protein in which this Tyr residue was substituted by Phe has been produced and isolated to homogeneity. It was spectroscopically analyzed by means of circular dichroism, fluorescence emission and NMR. Taken together, these results and those from enzyme characterization have revealed the essential role of the -OH group from the Tyr-48 phenolic ring in the cleavage of polymeric RNA substrates, including the ribosome-embedded 28S rRNA, the natural substrate of ribotoxins. Thus, the mutant protein does not degrade its natural ribosomal RNA substrate. However, it has been shown that this Y48F mutant still retains its ability to cleave a phosphodiester bond in a minimal substrate such as the dinucleoside phosphate ApA. The role of different alpha-sarcin residues within the enzyme reaction catalyzed by this protein is discussed.
Alvarez-García
Elisa
E
Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, E-28040 Madrid, Spain.
García-Ortega
Lucía
L
Verdún
Yolanda
Y
Bruix
Marta
M
Martínez del Pozo
Alvaro
A
Gavilanes
José G
JG
D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D010715:Phosphatidylglycerols / Q000737:chemistry; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012331:RNA, Fungal / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D050505:Mutant Proteins; D010715:Phosphatidylglycerols; D012331:RNA, Fungal; C014801:alpha-sarcin; D014443:Tyrosine; D010649:Phenylalanine; C002773:dimyristoylphosphatidylglycerol; D004722:Endoribonucleases