Rl. Deresiewicz et al., THE ROLE OF TYROSINE-114 IN THE ENZYMATIC-ACTIVITY OF THE SHIGA-LIKE TOXIN-I A-CHAIN, MGG. Molecular & general genetics, 241(3-4), 1993, pp. 467-473
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.