Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein.
Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.
Masip
Manuel
M
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, 28040-Madrid, Spain.
García-Ortega
Lucía
L
Olmo
Nieves
N
García-Mayoral
Maria Flor
MF
Pérez-Cañadillas
José Manuel
JM
Bruix
Marta
M
Oñaderra
Mercedes
M
Martínez del Pozo
Alvaro
A
Gavilanes
José G
JG
D019943:Amino Acid Substitution; D001665:Binding Sites; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005656:Fungal Proteins; D005658:Fungi / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D020871:RNA Stability; D012335:RNA, Ribosomal / Q000378:metabolism; D012208:Rhabdomyosarcoma / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D005656:Fungal Proteins; D012335:RNA, Ribosomal; C014801:alpha-sarcin; D004722:Endoribonucleases; D007930:Leucine