THE ROLE OF TYROSINE-114 IN THE ENZYMATIC-ACTIVITY OF THE SHIGA-LIKE TOXIN-I A-CHAIN

Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain p athogenic strains of Escherichia coli, is a member of a burgeoning fam ily of ribosome-inactivating proteins (RIPs), which share common struc tural and mechanistic features. The prototype of the group is the plan t toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subu nit of the ricin toxin. The model places three aromatic residues withi n the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, an d tryptophan 203. Here we present biochemical and biophysical data reg arding, the phenotypes of conservative point mutants of Slt-IA in whic h tyrosine 114 is altered. We used oligonucleotide-directed mutagenesi s to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutan t Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y114F was attenu ated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential acti vation of the mutants rather than local effects at the active site mig ht account for their diminished activity, we engineered the same mutat ions into a truncated slt-IA cassette that directs expression of a pro duct corresponding to the activated A, form of Slt-IA (wild-type-DELTA ). The same general relationships held: relative to wild type-DELTA, Y 114F-DELTA was attenuated about 7-fold, and Y114S-DELTA about 300-fold . Tryptic digestion profiles of the mutant proteins were similar to th ose of the corresponding wild-type, indicating that the amino acid sub stitutions had not caused major alterations in conformation. We conclu de that Y114 plays a significant role in the activity of Slt-IA, one w hich is quantitatively similar to that of Y77, and one which is predic ated on the presence of both its weakly acidic phenolic hydroxyl and i ts aromatic ring.