Kinetics of GLUT1-Catalyzed Movement of Glucose

As noted previously, a plot of the entry rate of glucose into erythrocytes versus external glucose concentration is not linear; rather, it is a curve that levels off at V_max at high external glucose concentrations (see Figure 15-5). The kinetics of the unidirectional transport of glucose (and other small molecules) from the outside of a cell inward via a uniporter can be described by the same type of equation used to describe a simple enzyme-catalyzed chemical reaction.

For simplicity, let’s assume that a substance S (say, glucose) is present initially only on the outside of the membrane. In this case, we can write

where K_m is the substance-transporter binding constant and V_max is the maximum transport rate of S into the cell. By a similar derivation used to arrive at the Michaelis-Menten equation in Chapter 3, we can derive the following expression for v, the transport rate for S into the cell:

For GLUT1 in the erythrocyte membrane, the K_m for glucose transport is 1.5 millimolar (mM); at this concentration roughly half the transporters with outward-facing binding sites would have a bound glucose. Blood glucose is normally 5 mM, or 0.9 g/L. At this concentration, the erythrocyte glucose transporter is functioning at 77 percent of the maximal rate V_max, as can be seen from Figure 15-5.

The kinetics of glucose transport are more complex and more revealing than this simple analysis suggests. For instance, if [¹⁴C]glucose is added to a suspension of erythrocytes whose intracellular glucose concentration is zero, the labeled glucose is transported inward at a particular initial rate proportional to the concentration of labeled glucose, as described by Equation 15-4. This initial rate of [¹⁴C]glucose transport is accelerated severalfold if unlabeled glucose is present inside the cells before addition of the labeled glucose. This unexpected experimental observation indicates that the slow (rate-determining) step in the inward transport of glucose is the change in GLUT1 from a conformation with an unoccupied inward-facing glucose-binding site to a conformation with an unoccupied outward-facing binding site (step 4 → step 5 in Figure 15-7). This conformational change is accelerated severalfold when an unlabeled glucose molecule binds to the inward-facing site and is transported outward. This result adds strong support to the conformational-change model of GLUT1 depicted in Figure 15-7.

Specificity and Structure of GLUT1

As noted above, the K_m for glucose transport by GLUT1 is 1.5 mM. The K_m for the nonbiological L-isomer of glucose is >3000 mM; thus at concentrations at which D-glucose is readily transported into the erythrocyte, L-glucose does not enter at a measurable rate. The isomeric sugars D-mannose and D-galactose, which differ from D-glucose in the configuration at only one carbon atom (see Figure 2-8), also are transported by GLUT1 at measurable rates. However, the K_m for D-mannose is 20 mM and for D-galactose is 30 mM, so that considerably higher concentrations of these substrates than of D-glucose are needed to half-saturate the transport reaction. Thus GLUT1 is quite specific, having a much higher affinity (indicated by a lower K_m) for the normal substrate D-glucose than for other substrates.

After glucose is transported into the erythrocyte, it is rapidly phosphorylated, forming glucose 6-phosphate, which cannot leave the cell (see Figure 16-3). Because this reaction is the first step in the metabolism of glucose, the intracellular concentration of free glucose does not increase as glucose is taken up by the cell. Consequently, the glucose concentration gradient across the membrane is maintained, as is the rate of glucose entry into the cell.

GLUT1 is an integral, transmembrane protein with a molecular weight of 45,000. It accounts for 2 percent of the protein in the plasma membrane of erythrocytes. Insertion of purified GLUT1 into artificial liposomes dramatically increases their permeability to D-glucose (see Figure 15-4). This artificial system exhibits all the properties of glucose entry into erythrocytes: in particular, D-glucose, D-mannose, and D-galactose are taken up, but L-glucose is not.

Amino acid sequence and biophysical studies on the glucose transporter indicate that it contains 12 α helices that span the phospholipid bilayer. Although the amino acid residues in the transmembrane α helices are predominantly hydrophobic, several helices bear amino acid residues (e.g., serine, threonine, asparagine, and glutamine) whose side chains can form hydrogen bonds with the hydroxyl groups on glucose. These residues are thought to form the inward-facing and outward-facing glucose-binding sites in the interior of the protein.