Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin

alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease, Three re sidues potentially involved in the mechanism of catalysis-histidine-50, glu tamic acid-96, and histidine-137-were changed to glutamine, Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant ( H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated t o homogeneity. These variants were spectroscopically (circular dichroism, f luorescence emission, and proton nuclear magnetic resonance) characterized, According to these results, the three-dimensional structure of these varia nts of alpha-sarcin was preserved; only very minor local changes were detec ted. Al the variants were inactive when assayed against either intact ribos omes or poly(A). The effect of pH on the ribonucleolytic activity of alpha- sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodieste r bond, but it did so to a lesser extent than did wild-type alpha-sarcin, T he results obtained are interpreted in terms of His137 and Glu96 as essenti al residues for the catalytic activity of alpha-sarcin (His137 as the gener al acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin. Proteins 1999;37: 474-484 . (C) 1999 Wiley-Liss, Inc.