Abstract
The effect of various galactose derivatives, substituted at C-6, on the phosphoenolpyruvate:beta-galactoside phosphotransferase system of Staphylococcus aureus was studied. Cells were grown by an improved procedure, which resulted in a 5- to 10-fold increase in cell yield. The four protein components of the system were separated. A membrane fraction containing negligible levels of the soluble components was prepared by alternate cycles of sonic treatment and differential centrifugation. The in vitro system reconstituted from these fractions was used to test the ability of the galactose derivatives to inhibit the phosphorylation of lactose analogs, under conditions where the membrane-bound component, Enzyme IIlac, was rate limiting. Derivaites in which the hydroxyl group of C-6 was missing, or replaced by a fluoro, O-methyl, or carboxyl group had no affinity for Enzyme IIlac, as judged by their inability to inhibit phosphorylation. Surprisingly, derivatives containing arylsulfonyl groups at C-6 were potent inhibitors; the O-tosyl compound has an apparent affinity five times that of galactose. The arylsulfonyl substitution in an absolute requirement; neither O-benzyl or O-methanesulfonyl derivatives were inhibitory. The specificity of the inhibition by tosyl derivatives parallels that of unsubstituted substrates; tosyl galactosides of the beta configuration were inhibitory, but those of the alpha configuration were not. The tosyl derivatives also strongly inhibited the uptake of lactose analogs into whole cells; the requirement for the arylsulfonyl moiety was again observed. The chemical analogy between the tosyl galactosides and possible intermediates in the transport-phosphorylation step catalyzed by Enzyme IIlac provides a possible explanation for the unexpected properties of these derivatives.
Highlights
The phosphorylation was shown to be mediated by a phosphoenolpyruvate-dependent phosphotransferase system [3,4,5,6], similar in many respects to the phosphotransferase system characterized in the gram-negative organism Escherichia coli
In this report we describe the inability of most C-6submitted galactose derivatives to inhibit the phosphotransferase system, and the unexpectedly high affinity of certain 6-arylsulfonyl-n-galactopyranosides for Enzyme IP, evidenced by their ability to inhibit both TMG
Galactoside, at a concentration of 4 mM, did not detectably inhibit TMG uptake. The failure of both Dfucose and 6-deoxy-Bfluorogalactose to inhibit TMG phosphorylation suggests that interaction with the hydrogen of the C-6 hydroxyl is an important element in the binding of substrates to Enzyme II’““, and is consistent with the reported inability of 6-deoxyglucose to inhibit the uptake of 2-deoxyglucose by Escherichia coli [10]
Summary
The effect of various galactose derivatives, substituted at C-6, on the phosphoenolpyruvate$galactoside phosphotransferase system of Stuphylococcus aureus was studied. The in vitro system reconstituted from these fractions was used to test the ability of the galactose derivatives to inhibit the phosphorylation of lactose analogs, under conditions where the membrane-bound component, Enzyme. Derivatives containing arylsulfonyl groups at C-6 were potent inhibitors; the 0-tosyl compound had an apparent affinity five times that of galactose. The tosyl derivatives strongly inhibited the uptake of lactose analogs into whole cells; the requirement for the arylsulfonyl moiety was again observed. The chemical analogy between the tosyl galactosides and possible intermediates in the transport-phosphorylation step catalyzed by Enzyme II’“’ provides a possible explanation for the unexpected properties of these derivatives. The lactose phosphotransferase system in S. aureus mediates a sequence of phosphoryl transfers according to the scheme below, detailed biochemical evidence for which has been presented previously (ll-13).*
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