BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

PART V. MOLECULAR PHYSIOLOGY

CHAPTER 36. MEMBRANE TRANSPORT

36.12. Active Transport of Certain Sugars Is Coupled to Their Phosphorylation

Symport is not the only type of pump mediating sugar transport. In some Bacteria, the accumulation of CARBOHYDRATES is coupled with their phosphorylation as they enter The Cell. For example, in many bacteria, glucose entering the cell is converted into glucose-6-phosphate. A distinctive feature of this transport type, known as group translocation, is that the solute is chemically modified in the process. The cell membrane is impermeable to phosphorylated sugars, which causes them to accumulate inside the bacterial cell.

Class="center">Table 36.1. Carbohydrates transported by the E. coli phosphotransferase system

Group translocation is best exemplified by the phosphotransferase system (PTS) discovered by Saul Roseman. A key feature of this system is that the phosphoryl group donor is phosphoenolpyruvate rather than ATP or any other nucleoside triphosphate. The overall reaction catalyzed by the phosphotransferase system is as follows:

Fig. 36.15. The proton gradient serves as an energy source for The transport of certain sugars and Amino Acids into bacterial Cells. The proton gradient is generated by electron flow in the Respiratory Chain

Four Proteins participate in this translocation process: HPr, enzyme I, enzyme II, and enzyme III. Enzyme II, an integral membrane protein, forms a transmembrane channel and catalyzes sugar phosphorylation. The phosphoryl group from phosphoenolpyruvate is not transferred directly to the sugar, but is first passed to enzyme I and then to a specific Histidine residue of a small thermostable protein, HPr (Fig. 36.16). The resulting phosphohistidine intermediate has a high phosphoryl group transfer potential, intermediate in magnitude between those of ATP and phosphoenolpyruvate. Next, the phosphoryl group is transferred from phosphorylated HPr to enzyme III, a peripheral membrane protein that interacts directly with the channel itself, i.e., enzyme II:

Fig. 36.16. Flow of phosphoryl groups from phosphoenolpyruvate to the sugar transported across the membrane by the phosphotransferase system

The final step is The transfer of the phosphoryl group from enzyme III to the sugar being transported (Fig. 36.17). A functionally active enzyme complex has been successfully reconstructed from these four purified proteins.

Fig. 36.17. Proposed mechanism of group translocation by the phosphotransferase system

Some Proteins of the phosphotransferase system are specific, whereas others are not. For instance, HPr and enzyme I, which are soluble cytosolic proteins, participate in the transport of all sugars translocated by this system. Conversely, Enzymes II and III exhibit Specificity toward particular sugars. For example, distinct enzymes II and III are involved in the transport of glucose, lactose, and fructose. Genetic studies have yielded identical findings: mutants defective in HPr or enzyme I fail to transport A wide variety of different sugars, whereas mutants defective in the synthesis of enzymes II and III are unable to transport only a specific sugar. Enzyme III does not participate in the translocation of hexitols, such as galactitol; in this case, the phosphoryl group is transferred directly from HPr to the carbohydrate.

Why is the phosphotransferase system structurally much more complex than other carriers, such as the lactose permease? It seems likely that the phosphotransferase system not only mediates sugar transport but also performs regulatory Functions. Excessive uptake of a single carbohydrate via the phosphotransferase system strongly inhibits the Active Transport of other sugars by different carriers. This inhibition is apparently mediated by changes in cAMP levels; specifically, an increase in the concentration of the sugar accumulated by the phosphotransferase system leads to a decrease in cAMP production (Fig. 36.18). As a result, METABOLISM/31.html">Transcription of several inducible operons is halted. Recall that the expression of inducible operons such as lac and gal increases significantly upon the binding of the cAMP-CRP complex to specific promoter sites (Section 28.6). Therefore, the phosphotransferase system regulates carbon source utilization.

Fig. 36.18. The phosphotransferase system-transported sugar α-methylglucoside inhibits cAMP production

36.13. Transport Antibiotics Increase Membrane Ion Permeability

A number of microorganisms synthesize low-molecular-weight compounds that render membranes permeable to specific ions. These small molecules, known as transport Antibiotics, have proven to be valuable tools for experimental research, particularly in investigating ion-binding mechanisms. For example, valinomycin uncouples Oxidative Phosphorylation in Mitochondria by increasing their permeability to K+: in the presence of valinomycin, mitochondria use the energy generated by electron transport not for ATP synthesis, but for K⁺ accumulation. Valinomycin has a cyclic Structure composed of a repeating sequence of four different residues (A, B, C, and D) (Fig. 36.19). These four types of residues are connected by alternating ester and peptide bonds.

Fig. 36.19. Valinomycin has a periodic cyclic structure consisting of residues of L-lactate (A), L-valine (B), D-hydroxyisovalerate (C), and D-valine (D)

Another well-studied transport antibiotic is gramicidin A (Fig. 36.20). It is an open-chain polypeptide consisting of 15 amino acid residues. A notable feature of gramicidin A's structure is the alternation of D- and L-amino acids. Furthermore, the N- and C-termini of the polypeptide are modified. As will be discussed below, the transport of ions by gramicidin A and valinomycin occurs in entirely different ways.

Fig. 36.20. Structure of gramicidin A

The Mechanism of ion translocation by these antibiotics is conveniently investigated using well-characterized model systems such as phospholipid vesicles (Section 10.6) and planar lipid bilayers (Section 10.6). The experiments utilize vesicles containing a radioactive ion, such as ⁴²K⁺; these are prepared by sonicating membranes in the presence of this ion and subsequently removing the external ⁴²K⁺ by Gel filtration. The rate of radioactive ion efflux from the vesicles is then compared in the presence and absence of the antibiotic. Another approach to studying the ion permeability of bilayer membranes involves measuring electrical parameters such as membrane resistance and Membrane Potential. For example, the resistance of a bilayer membrane to K⁺ in the presence of 10^-7 M valinomycin or 10^-9 M gramicidin drops by a factor of over 10,000 at a KCl concentration gradient across the membrane of 0.02 M.

36.14. Transport Antibiotics Function Either as Mobile Carriers or as Channel Formers

There are two completely distinct mechanisms by which transport antibiotics affect membrane ion permeability (Fig. 36.21). Some antibiotics (such as gramicidin A) form a channel that spans the membrane. Ions enter such a channel on one side of the membrane, diffuse through it, and exit on the other side. The stimulation of ion transport via this mechanism is not coupled to the movement of the channel-forming antibiotic itself. Antibiotics of another group (such as valinomycin) function as carriers that shuttle ions across the Hydrophobic core of the membrane. The activity of these transport antibiotics is coupled to their own diffusion.

Fig. 36.21. Schematic illustration of the differences between channel-forming transport antibiotics and mobile carriers. All known biological transport proteins belong to the category of channel formers

In experiments, mobile carriers and channel formers can be distinguished in the following manner. The Temperature dependence of the ionic conductance of an artificial lipid bilayer containing the transport antibiotic is measured over a temperature range that encompasses the lipid phase transition; within this range, the hydrocarbon interior of the membrane goes from a nearly solid to a completely fluid state. A channel former mediates Ion transport across the membrane without undergoing translocation itself. Consequently, the solidification of the hydrocarbon layer will not significantly affect its ability to transport ions. The situation is quite different for a mobile ion carrier, which must diffuse across the membrane's hydrocarbon core to be active: its efficiency should decrease markedly when the hydrocarbon layer solidifies. Experimental data with valinomycin and gramicidin A indeed reveal a clear distinction between these two mechanisms (Fig. 36.22). The permeability of a valinomycin-containing bilayer increases more than 1,000-fold upon fluidization. In contrast, the transport activity of gramicidin A is virtually unaffected by the phase transition of the membrane to the fluid state.

Fig. 36.22. Effect of temperature on the conductance of lipid bilayer membranes, one containing a channel-forming transport antibiotic and the other a mobile carrier transport antibiotic

It is important to emphasize that all currently known natural transport systems function as channels.

As discussed above (Section 10.14), the translocation of integral Membrane Proteins from one membrane surface to the other (transverse diffusion) is extremely slow or absent altogether. Consequently, they cannot act as mobile carriers.