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-a rabinose (AraE) and D-galactose (GalP) symporters from Escherichia col i and to the human erythrocyte glucose transporter (GLUT1) have been i nvestigated by exploiting the changes in protein fluorescence that occ ur upon binding the ligand. Steady-state measurements yielded K-d valu es of 1.1, 1.9 and 0.14 mu M for the AraE, GalP and GLUT1 proteins, re spectively. The association and dissociation rate constants for the bi nding of cytochalasin B have been determined by stopped-flow spectrosc opy. In each case, the apparent K-d was calculated from the correspond ing 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 cy tochalasin B: [GRAPHICS] The transporter is proposed to alternate betw een two different conformational forms (T-1 and T-2), with cytochalasi n B binding only to the T-2 conformation, to induce a further conforma tional transition of the transporter to the T-3 form. The values for t he overall dissociation constants show that the T-1 conformation is fa voured by AraE and GalP in the absence of ligands, but the T-2 conform ation is favoured by GLUT1. Thus, the binding of cytochalasin B to GLU T1 alters the equilibrium towards the T-3(CB) conformational state, pr oducing the observed tight binding, in contrast to the changes in the equilibrium observed with the binding of cytochalasin B to AraE and Ga lP. A thermodynamic analysis of these conformational transitions has b een performed. The T-1 and T-2 conformations may represent transporter states in which the binding site is facing outwards and inwards, resp ectively.