Different sidedness of functionally homologous essential thiols in two membrane-bound phosphotransferase enzymes of Escherichia coli detected by permeant and nonpermeant thiol reagents.

The membrane-bound Enzyme IIbgl and IIglc are both inactivated in vivo by the sulfhydryl reagent N-ethylmaleimide. The former is also inhibited by the hydrophilic sulfhydryl reagents p-chloromercuribenzoic acid and p-mercuriphenylsulfonic acid, while the latter is resistant to these reagents. However, inhibition of this enzyme is observed after impairment, either transient or permanent, of the permeability barrier of bacterial envelopes. Since p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid are able to cross the outer membrane of Escherichia coli, their failure to inhibit in vivo Enzyme IIglc must be due to their inability to cross the inner membrane of the bacteria. It would therefore appear that sensitive thiol group(s) of Enzyme IIglc and Enzyme IIbgl, in spite of their functional similarity, exhibit opposite orientation within the cytoplasmic membrane, the first enzyme having an -SH group accessible from the outer surface of the membrane, while the second has an -SH group accessible from the inner surface of the membrane. The present results strengthen the view that these two enzymes have in asymmetric orientation within the membrane as already suggested by their vectorial function.