UNMASKING OF AN ESSENTIAL THIOL DURING FUNCTION OF THE MEMBRANE-BOUND ENZYME-II OF THE PHOSPHOENOLPYRUVATE BETA-GLUCOSIDE PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme IIbgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme IIglc. It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme IIbglc and p-nitrophenyl-β-d-glucoside only sensitizes enzyme IIbgl towards N-ethylmaleimide inactivation. The inactivation of enzyme IIbgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of p-nitrophenyl-β-d-glucoside phosphorylation. Both results suggest that the inactivator resistent form of enzyme IIbgl is an energized form of the enzyme. © 1979.