THE KINETICS AND THERMODYNAMICS OF THE BINDING OF CYTOCHALASIN-B TO SUGAR TRANSPORTERS

The kinetics of the binding of cytochalasin B to the proton-linked L-arabinose (AraE) and D-galactose (GalP) symporters from Escherichia coli and to the human erythrocyte glucose transporter (GLUT1) have been investigated by exploiting the changes in protein fluorescence that occur upon binding the ligand. Steady-state measurements yielded K-d values of 1.1, 1.9 and 0.14 mu M for the AraE, GalP and GLUT1 proteins, respectively. The association and dissociation rate constants for the binding of cytochalasin B have been determined by stopped-flow spectroscopy. In each case, the apparent K-d was calculated from the corresponding rate constants, yielding values of 1.5, 0.4 and 1.6 mu M for AraE, GalP and GLUT1, respectively. The differences between these apparent K-d values and those measured by fluorescence titration is interpreted in terms of the following three step mechanism where CB represents cytochalasin B: [GRAPHICS] The transporter is proposed to alternate between two different conformational forms (T-1 and T-2), with cytochalasin B binding only to the T-2 conformation, to induce a further conformational transition of the transporter to the T-3 form. The values for the overall dissociation constants show that the T-1 conformation is favoured by AraE and GalP in the absence of ligands, but the T-2 conformation is favoured by GLUT1. Thus, the binding of cytochalasin B to GLUT1 alters the equilibrium towards the T-3(CB) conformational state, producing the observed tight binding, in contrast to the changes in the equilibrium observed with the binding of cytochalasin B to AraE and GalP. A thermodynamic analysis of these conformational transitions has been performed. The T-1 and T-2 conformations may represent transporter states in which the binding site is facing outwards and inwards, respectively.